Periodic Reporting for period 4 - BIO-H-BORROW (Biocatalytic Amine Synthesis via Hydrogen Borrowing)
Période du rapport: 2021-12-01 au 2023-05-31
Nature generates amines (e.g. alkaloids, amino acids) via multienzyme biosynthetic pathways and in doing so creates some of the most complex natural products that have been discovered to date (e.g. morphine, vinblastine, reserpine). These biosynthetic pathways are truly remarkable in the way in which many enzymes are able to collectively process substrates in an orchestrated assembly line manner with no use of protecting groups and minimal side reactions. Despite our much deeper understanding of how these biosynthetic pathways operate, in terms of the substrates and enzymes involved, only recently have scientists begun to assemble these pathways in host systems for eventual scale-up and production of opiates.
Most synthetic amines are produced using abiological methods, particularly where cost and scale of operation are important. The majority of these chemical processes for amine production involve the use of high temperatures, pressures and expensive metal catalysts. In addition, the precursors for amine manufacture are largely derived from the petrochemical industry and hence are non-sustainable. Clearly a major challenge going forward is to try to recreate these cascade processes, both in vitro and in vivo, particularly for the production of non-natural synthetic amines.
The overall aim of the BIO-H-BORROW project is to develop a new biocatalytic approach for amine synthesis in which alcohols are used as universal substrates, ammonia is the source of the amine in the product, and the only by-product of the reaction is water. Initial work focused on characterisation and engineering of suitable enzymes and development of supporting analytical methodologies.
Most synthetic amines are produced using abiological methods, particularly where cost and scale of operation are important. The majority of these chemical processes for amine production involve the use of high temperatures, pressures and expensive metal catalysts. In addition, the precursors for amine manufacture are largely derived from the petrochemical industry and hence are non-sustainable. Clearly a major challenge going forward is to try to recreate these cascade processes, both in vitro and in vivo, particularly for the production of non-natural synthetic amines.
The overall aim of the BIO-H-BORROW project is to develop a new biocatalytic approach for amine synthesis in which alcohols are used as universal substrates, ammonia is the source of the amine in the product, and the only by-product of the reaction is water. Initial work focused on characterisation and engineering of suitable enzymes and development of supporting analytical methodologies.
The overall objective BIO-H-BORROW was to significantly increase the range and diversity of biocatalysts available for chiral amine synthesis, particularly using ammonia as the source of the amine group and to demonstrate this through the synthesis of pharmaceutically relevant molecules and compounds.
In order to achieve this objective we adopted a number of different strategies the focussed on the key stages of the synthetic regimen for the production of amines from alcohols. These stages included: 1) generation of novel panels of imine reductases (IREDS) and reductive aminases (RedAms) with enzymes selected from metagenomic sequence data. 2) engineering of existing biocatalysts to expand the substrate scope and improve activity and selectivity, e.g. for amine dehydrogenases (AmDHs), and 3) the development of high throughput screening methods to enable the characterisation of large panels of biocatalysts. It was through this process of enzyme discovery and engineering that we were able to identify novel IREDs and RedAms able to use ammonia as the source of the amine group, the first such example of this reaction. This lead us to refine our proposal for the mechanism by which these enzymes catalyse reductive amination.
Regarding the central idea of BIO-H-BORROW, namely the concept of constructing multi-enzyme cascades to allow redox neutral conversion of alcohols to amines we adopted a broad approach to this challenge. Work in our group had shown that the combined use of nicotinamide dependent oxidoreductases (e.g. combination of alcohol DHs with amine DHs) to convert alcohols to amines could be achieved but that there were some limitations regarding overall conversion due to equilibrium issues. We therefore developed a new approach is which the conversion of alcohols to ketones/aldehydes was catalysed by an oxidase enzyme. The advantage of this method is that oxidase reactions are irreversible hence removing problems with the overall equilibrium. In this respect we successfully engineered a choline oxidase enzyme for broad substrate scope and showed that this enzyme could be combined in a cascade process with IREDs and RedAms to convert alcohols to amines in one-pot.
Importantly, through the work carried out within BIO-H-BORROW we also demonstrated that our new panel of IREDs and RedAms could be applied to the synthesis of key pharmaceutical building blocks which led to rapid uptake of our technology by industry. The IRED and RedAm panel is available to purchase through the biocatalyst supply company Prozomix.
In parallel with the development of enzyme cascades for chiral amine synthesis we felt that it was important to address the issue of providing tools for the wider synthetic community to aid in the implementation of biocatalysis in synthetic route planning. This thinking ultimately led to the development of RetroBioCat (www.retrobiocat.com) an open access collection of tools for in silico biocatalytic cascade design. RetroBioCat has been widely adopted by the synthesis community in both academe and industry and has resulted in the formation of a start-up company Disyn Biotec (www.disynbiotec.com) which make available commercial licences for RetroBioCat.
In order to achieve this objective we adopted a number of different strategies the focussed on the key stages of the synthetic regimen for the production of amines from alcohols. These stages included: 1) generation of novel panels of imine reductases (IREDS) and reductive aminases (RedAms) with enzymes selected from metagenomic sequence data. 2) engineering of existing biocatalysts to expand the substrate scope and improve activity and selectivity, e.g. for amine dehydrogenases (AmDHs), and 3) the development of high throughput screening methods to enable the characterisation of large panels of biocatalysts. It was through this process of enzyme discovery and engineering that we were able to identify novel IREDs and RedAms able to use ammonia as the source of the amine group, the first such example of this reaction. This lead us to refine our proposal for the mechanism by which these enzymes catalyse reductive amination.
Regarding the central idea of BIO-H-BORROW, namely the concept of constructing multi-enzyme cascades to allow redox neutral conversion of alcohols to amines we adopted a broad approach to this challenge. Work in our group had shown that the combined use of nicotinamide dependent oxidoreductases (e.g. combination of alcohol DHs with amine DHs) to convert alcohols to amines could be achieved but that there were some limitations regarding overall conversion due to equilibrium issues. We therefore developed a new approach is which the conversion of alcohols to ketones/aldehydes was catalysed by an oxidase enzyme. The advantage of this method is that oxidase reactions are irreversible hence removing problems with the overall equilibrium. In this respect we successfully engineered a choline oxidase enzyme for broad substrate scope and showed that this enzyme could be combined in a cascade process with IREDs and RedAms to convert alcohols to amines in one-pot.
Importantly, through the work carried out within BIO-H-BORROW we also demonstrated that our new panel of IREDs and RedAms could be applied to the synthesis of key pharmaceutical building blocks which led to rapid uptake of our technology by industry. The IRED and RedAm panel is available to purchase through the biocatalyst supply company Prozomix.
In parallel with the development of enzyme cascades for chiral amine synthesis we felt that it was important to address the issue of providing tools for the wider synthetic community to aid in the implementation of biocatalysis in synthetic route planning. This thinking ultimately led to the development of RetroBioCat (www.retrobiocat.com) an open access collection of tools for in silico biocatalytic cascade design. RetroBioCat has been widely adopted by the synthesis community in both academe and industry and has resulted in the formation of a start-up company Disyn Biotec (www.disynbiotec.com) which make available commercial licences for RetroBioCat.
Prior to commencing work on this ERC advanced grant Dr. R. HEATH worked on the development of a Choline Oxidase enzyme. This researcher was funded by European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 613849 supporting the project BIOOX. Choline oxidase, in conjunction with RedAm, was initially used as a comparison for the Hydrogen Borrowing technology and has highlighted the limitations of the current state of the art. While the Hydrogen Borrowing system allows for redox neutral amine synthesis, the reversibility of both the ADH and AspRedAm enzyme catalysed reactions leads to conversions that ultimately are limited by reaction thermodynamics. This leads to a requirement for a large excess of amine substrate and is more pronounced in the synthesis of secondary amines, where the equilibrium is less favourable. These issues are not obvious at analytical reaction scale reaction but present difficulties when the reaction is scaled. Choline oxidase catalyses an irreversible alcohol oxidation reaction and as such there are no issues with the thermodynamic equilibrium position between the alcohol and carbonyl intermediate and thus significantly higher conversions can be achieved. We have demonstrated a cascade involving a variant of Choline Oxidase coupled with AspRedAm by screening a panel of eight alcohols with five different amines.
Through the BIO-H-BORROW research programme we identified a number of new biocatalytic reactions that were otherwise unknown. These include novel IREDs and RedAms able to use ammonia as the source of the amine group, the identification and characterisation of a new family of enzymes which we termed ene imine reductases (EneIREDs). Remarkably these enzymes were found to be able to catalyse three distinct reactions within a single active-site. It is these new discoveries that have helped us push the boundaries of what is possible with already established enzymes. This has further strengthened the position of biocatalysts as the go to method for sustainable manufacture of amines from alcohols.
Through the BIO-H-BORROW research programme we identified a number of new biocatalytic reactions that were otherwise unknown. These include novel IREDs and RedAms able to use ammonia as the source of the amine group, the identification and characterisation of a new family of enzymes which we termed ene imine reductases (EneIREDs). Remarkably these enzymes were found to be able to catalyse three distinct reactions within a single active-site. It is these new discoveries that have helped us push the boundaries of what is possible with already established enzymes. This has further strengthened the position of biocatalysts as the go to method for sustainable manufacture of amines from alcohols.