Building synthetic enzyme fusions for chiral molecule production.
Lead Research Organisation:
University College London
Department Name: Structural Molecular Biology
Abstract
Strategic Research Priority: World Class Underpinning Research
Optically active amines play an important role as bioactive compounds in pharmaceutical and chemical industries. Because classical chemical approaches are complex and expensive, biocatalytic routes are desirable. One route for their bio-production involves conversion of alcohols to amines. Currently, there are no known enzymes that can convert alcohols directly to amines, however a two-step transformation is possible via alcohol oxidation and a subsequent reductive amination or transamination.
In enzyme cascades, reaction engineering is necessary to ensure flux towards the products, and to avoid accumulation of, often, reactive intermediates. In contrast, naturally occurring multifunctional enzymes have evolved to keep reactive substrates within enzyme complexes until key transformations have been carried out. In vivo, multifunctional enzymes are thought to confer selective advantage via increased reaction rates and chemical stability or prevention of toxicity from reactive intermediates.
The aim of this project was to characterise a natural fusion between a transaminase and a dehydrogenase, protein PP_2782, in Pseudomonas putida KT2440 (a non-pathogenic soil bacterium that has a diverse range of amino and fatty acid metabolic pathways), in order to exploit its biosynthetic abilities for novel enzyme fusion engineering. Using this natural fusion as a guide template, and to enable the synthesis of novel bioactive compounds that are challenging to produce chemically, a number of synthetic fusions comprising well-characterised enzymes was engineered and tested.
Optically active amines play an important role as bioactive compounds in pharmaceutical and chemical industries. Because classical chemical approaches are complex and expensive, biocatalytic routes are desirable. One route for their bio-production involves conversion of alcohols to amines. Currently, there are no known enzymes that can convert alcohols directly to amines, however a two-step transformation is possible via alcohol oxidation and a subsequent reductive amination or transamination.
In enzyme cascades, reaction engineering is necessary to ensure flux towards the products, and to avoid accumulation of, often, reactive intermediates. In contrast, naturally occurring multifunctional enzymes have evolved to keep reactive substrates within enzyme complexes until key transformations have been carried out. In vivo, multifunctional enzymes are thought to confer selective advantage via increased reaction rates and chemical stability or prevention of toxicity from reactive intermediates.
The aim of this project was to characterise a natural fusion between a transaminase and a dehydrogenase, protein PP_2782, in Pseudomonas putida KT2440 (a non-pathogenic soil bacterium that has a diverse range of amino and fatty acid metabolic pathways), in order to exploit its biosynthetic abilities for novel enzyme fusion engineering. Using this natural fusion as a guide template, and to enable the synthesis of novel bioactive compounds that are challenging to produce chemically, a number of synthetic fusions comprising well-characterised enzymes was engineered and tested.
Organisations
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M009513/1 | 01/10/2015 | 31/03/2024 | |||
| 1618783 | Studentship | BB/M009513/1 | 01/10/2015 | 30/09/2022 | Luba Prout |
| Description | This work sought to design and produce synthetic bifunctional catalysts that are inspired by nature, in order to enhance the performance of artificial pathways developed for the biosynthesis of complex amine compounds. Ultimately, the goal of this project has been achieved, whereby the natural enzyme fusion PP_2782 from Pseudomonas putida KT2440 as well as three of its thermophilic homologs were partially characterised, and novel synthetic fusions comprising 1) Chromobacterium violaceum DSM 30191 ?-transaminase, and (2) Equus caballus alcohol dehydrogenase, (3) Geobacillus stearothermophilus 22 alcohol dehydrogenase, and (4) Thalictrum flavum subsp. glaucum (S)-norcoclaurine synthase enzymes were engineered and shown to have a superior activity to cascades employing unfused enzymes. The investigation of the natural fusions provided the first detailed account of fusion enzymes of this kind identifying both the substrate range and scope of enzyme activity in different conditions. Overall, this project contributed to the acceleration of the development of novel 'green chemistry' approaches for the production of valuable biochemical materials. |
| Exploitation Route | Characterisation and analysis of natural transaminase-oxidoreductase fusions provide the basis for future academic work pursuing evolution of naturally-derived bifunctional catalysts and seeking to obtain the fusion enzyme structure. The engineered synthetic fusions produced by this work can facilitate the improvement of amine biosynthesis processes in complex pathways and can be utilised in industrial biocatalysis. |
| Sectors | Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | Cost Action (BioTrans 2017 meeting travel expenses) |
| Amount | € 550 (EUR) |
| Funding ID | CM1303 |
| Organisation | European Cooperation in Science and Technology (COST) |
| Sector | Public |
| Country | Belgium |
| Start | 07/2017 |
| End | 07/2017 |
| Description | Cost Action (Novel Enzymes 2016 meeting travel expenses) |
| Amount | € 536 (EUR) |
| Funding ID | CM1303 |
| Organisation | European Cooperation in Science and Technology (COST) |
| Sector | Public |
| Country | Belgium |
| Start | 10/2016 |
| End | 10/2016 |
| Title | Python-based genomic sequence visualiser for multiple genes |
| Description | The developed tool is a Python-based script written to visualise the genomic environment of one or more genes in order to enable user-controlled analyses and comparisons of gene clusters. |
| Type Of Material | Computer model/algorithm |
| Year Produced | 2018 |
| Provided To Others? | No |
| Impact | The tool was used to analyse the genomic environment of proteins/genes of interest (homologs) in various bacterial species in order to identify individual open reading frames and gene clusters that could potentially be involved in secondary metabolite biosynthesis. |
| Description | LIDo PhD programme 3i showcase at Genesis Fringe 2017 |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Industry/Business |
| Results and Impact | Attended a networking event presenting research in the form of a poster. Various business industry professionals enquired about potential future applications of my research. |
| Year(s) Of Engagement Activity | 2017 |
| Description | LIDo outreach symposium at GSK |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Industry/Business |
| Results and Impact | Attended a networking event presenting research in the form of a poster. Various industry professionals enquired about the work. |
| Year(s) Of Engagement Activity | 2018 |
| Description | LIDo retreat 2017 |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Presented my project in a three-minute thesis (3MT) format to an audience of PhD students with different bioscience backgrounds. This led to various discussions about the project with some of the PhD students. |
| Year(s) Of Engagement Activity | 2017 |
| Description | LIDo retreat 2018 |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Presented the project as a poster to an audience of PhD researchers with different multidisciplinary bioscience backgrounds. A number of PhD students enquired about my research and its application prospects. |
| Year(s) Of Engagement Activity | 2018 |
| Description | London Synbio Network Meetings |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | London Synbio Network (which started at UCL) facilitates Technical meet ups for PhD students and post docs. The purpose of these meetings is to share, discuss and troubleshoot projects. I attended several such meetings and shared my research with local community at UCL. |
| Year(s) Of Engagement Activity | 2017,2018 |
| Description | Presentation to Birkbeck College undergraduates |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Undergraduate students |
| Results and Impact | Presented my research to undergraduate students at Birkbeck. This resulted in a number of students becoming interested in and enquiring about research summer placements. |
| Year(s) Of Engagement Activity | 2017 |
| Description | UCL Synthetic Biology Showcase 2017 |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | Presented the project to an international audience of synthetic biology researchers and scientists with related background. Questions and comments helped to shape some aspects of the work. |
| Year(s) Of Engagement Activity | 2017 |
| Description | UCL Synthetic Biology Showcase 2018 |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | Presented my project as a poster to an international audience with multidisciplinary bioscience background. A number researchers enquired about my methodology, research and its future applications. |
| Year(s) Of Engagement Activity | 2018 |