Synthetic Biology Pathways to Isoquinoline Alkaloids
Lead Research Organisation:
University College London
Department Name: Structural Molecular Biology
Abstract
For thousands of years mankind has been using plants and medicinal compounds from plants for treating pain, diseases, infections and also for recreational purposes. Many of these plant derived compounds have also found their way into modern drug use for example morphine and codeine for pain relief, atropine to increase heart rate and for irritable bowel syndrome, vinblastine to treat cancers and quinine for malaria. There are over 14,000 alkaloid chemical structures known and these represent a wealth of potential biological activity. However many of these alkaloids occur in small amounts or in plants that are rare or only grow in parts of the world that are difficult to access. The pathways that plants use to synthesise alkaloids are often long with many biosynthetic steps. There are some key intermediate compounds that are common to many plant alkaloid pathways and the complexity of alkaloid structures comes from the different later steps in the pathways that different plants have. It would be of great benefit to be able to make some of these key intermediate alkaloid compounds in a more controlled way and to be able to modify these alkaloid structures using enzymes or chemistry. In this way novel biologically active drugs could be found. The pathways for the synthesis of several alkaloids have begun to be determined but many of the steps for some of the more complex alkaloids are still unknown. In the last few years researchers have begun to isolate the enzymes (and their genes) for some of the key steps in the synthesis of some alkaloids. Some of these enzymes carry out very important steps such as the joining together of rather simple compounds derived from simple amino acids. We intend to use the techniques of whole gene synthesis, synthetic biology and pathway design to make some of these key alkaloid synthesising enzymes and put them into a laboratory bacterium. In this way we can combine different plant based and bacterial enzymes and build synthetic pathways for the creation of new chemical compounds that could be screened for useful medicinal activities. By having the pathways in bacteria we would be able to generate them in a reproducible way using a renewable resource and not be dependant on the growth of difficult or rare plants. In addition, extensive chemical transformations using methods with high environmental impact would not be required which are currently used to synthesise such isoquinolines.
Technical Summary
We aim to construct recombinant Escherichia coli carrying synthetic engineered pathways for the production of alkaloids of the isoquinolines class. To do this we will use synthetic genes for the main coupling enzyme from the benzylisoquinoline (BIA) pathway, the norcoclaurine synthase (NCS). There are two types of NCS and these genes will be made synthetically and expressed in E. coli. The NCS joins dopamine to 4-hydroxyphenylacetaldehyde to make (S)-norcoclaurine. The substrate specificity of these two classes of wild type enzyme will be determined towards a range of aromatic amines such as substituted dopamines and tryptamines. The specificity towards a range of aldehydes including modified phenylacetaldehyes (4-OH, 3-OH, 3,4-diOH, halogen, amino etc), indoleacetaldehyde and aliphatic or cyclic aldehydes will be established. The NCS enzymes will be tested and used as stand alone enzymes for the coupling of the amine and aldehyde. Mutant NCS enzymes will be made with the ability to accept different and non-natural amines and aldehydes. The structures of the compounds formed by the native and mutant NCS enzymes will be determined using mass spectrometry and NMR spectroscopy. We will also construct the pathways which will enable E. coli to synthesise the dopamine and 4-hydroxyphenylacetaldehyde for the NCS and to combine these pathways to make strains which will make (S)-norcoclaurine de novo. Using synthetic genes for many of these feeder pathway components will allow different aromatic aldehydes to be made in vivo. E. coli expressing only one or the other pathway for the NCS substrates will allow non-natural amines or aldehydes to be fed to strains which carry pathways for the other NCS substrate and the NCS. Cytochrome P450 enzymes systems which have previously been constructed will be expressed together with NCS enzymes and the feeder pathways plus NCS. In this way we aim to build strains which can make diverse alkaloids of the isoquinoline class.
Organisations
People |
ORCID iD |
John Ward (Principal Investigator) | |
Helen Hailes (Co-Investigator) |
Publications
Pesnot T
(2012)
The Catalytic Potential of Coptis japonica NCS2 Revealed - Development and Utilisation of a Fluorescamine-Based Assay
in Advanced Synthesis & Catalysis
Bommer M
(2016)
Micromolar colorimetric detection of 2-hydroxy ketones with the water-soluble tetrazolium WST-1.
in Analytical biochemistry
Lechner H
(2018)
Library of Norcoclaurine Synthases and Their Immobilization for Biocatalytic Transformations.
in Biotechnology journal
Pesnot T
(2011)
Phosphate mediated biomimetic synthesis of tetrahydroisoquinoline alkaloids.
in Chemical communications (Cambridge, England)
Lichman B
(2015)
One-pot triangular chemoenzymatic cascades for the syntheses of chiral alkaloids from dopamine
in Green Chemistry
Pesnot T
(2017)
One-Pot Phosphate-Mediated Synthesis of Novel 1,3,5-Trisubstituted Pyridinium Salts: A New Family of S. aureus Inhibitors.
in Molecules (Basel, Switzerland)
Lichman BR
(2015)
'Dopamine-first' mechanism enables the rational engineering of the norcoclaurine synthase aldehyde activity profile.
in The FEBS journal
Guzman JD
(2015)
Tetrahydroisoquinolines affect the whole-cell phenotype of Mycobacterium tuberculosis by inhibiting the ATP-dependent MurE ligase.
in The Journal of antimicrobial chemotherapy
Description | Synthetic plant norcoclaurine synthase enzymes and mutants of these enzymes that can synthesise novel bioactive benzylisoquinoline alkaloids have been developed. Together with a fluorescence assay developed in the project we now have the means to rapidly make, screen and develop novel compounds. Two new chemical synthetic methods for making benzylisoquinoline and pyridinium alkaloids have been developed and one of these methods is being patented. Some of the compounds from these new synthetic methods have antimicrobial activity against bacteria such as Staphylococcus aureus and Mycobacterium tuberculosis and are the subject of a second patent. A new mechanism for the norcoclaurine synthase (NCS) enzyme has been proposed and has been published. The new mechanism leads to the development of better NCS enzymes, which we have identified, for making new bioactive compounds. |
Exploitation Route | The generation of new classes of compounds using the methods that we have developed could be used for making new pharmaceutical drugs for antimicrobials and for other pharmaceutical research and development areas. Some members of the new BIAs made by the phosphate reaction are able to inhibit Mycobacterium bovis and Mycobacterium tuberculosis. In a collaboration with researchers at Birkbeck College we have shown good anti-mycobacterial activity and low activity to mouse macrophages showing that these new reagents may have utility in treating TB infections. A draft publication has been prepared and we are seeking patent protection prior to submission. A second phosphate based reaction was discovered as a side reaction when the phosphate reaction is carried out at a high temperature. This reaction produces different non-BIA structures that are novel and we have tested the antimicrobial activity and established they have antibacterial activity towards Gram positive bacteria such as Staphylococcus aureus, Bacillus subtilis, Staph. carnosus and Micrococcus luteus. We have a draft publication and are currently seeking patent protection for the chemical method, new reagents and bioactivities of the compounds. |
Sectors | Agriculture, Food and Drink,Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
URL | http://www.ucl.ac.uk/biochemeng/people/academic/ward-j |
Description | We describe our research findings in our websites and to visiting school students. |
First Year Of Impact | 2016 |
Sector | Agriculture, Food and Drink,Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Impact Types | Societal |
Description | 17-ERACoBioTech Methyl Transferases for the Functional Diversification of Bioactives: BioDiMet |
Amount | £1,911,000 (GBP) |
Funding ID | BB/R021643/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 04/2018 |
End | 04/2022 |
Description | Enzyme Cascades and Synthetic Biology Routes to Non-Natural Alkaloids |
Amount | £457,840 (GBP) |
Funding ID | BB/N01877X/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 10/2016 |
End | 12/2019 |
Description | Innovate UK Health and Life Sciences Round 1 |
Amount | £585,195 (GBP) |
Funding ID | 84474-536277 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 05/2017 |
End | 04/2019 |
Title | Refining methods for multiple enzyme expression for developing enzyme cascades. |
Description | The need for building multiple enzyme cascades or short pathways is central to several areas of biocatalysts and synthetic biology. We have used the developments made in this grant and extended these to the research in several other grant funded areas. We can use comparable plasmids to co-express two or more enzymes in the same cell and we have been building short operons where all the genes are expressed from a single promoter on the same mRNA. With the comparable plasmid approach we can adjust the amount of each of the expressed enzymes by choosing plasmid replicons of different copy numbers as well as using different strength promoters. With the operon constructions we can ensure that all the enzymes are expressed at the same time and largely to the same levels. |
Type Of Material | Technology assay or reagent |
Provided To Others? | Yes |
Impact | The application of the methods and concepts described above has allowed us to create stable expression strains that don't need any antibiotic in continuous cultures. We have been able to balance the three different enzymes needed for functional cytochrome P450 expression. The technology is central now to several areas of our research for the synthesis of chiral small molecules for alkaloids, chiral amines and methyl transferases where several enzymes are needed to build the complex final compound. In several cases this can compete very favourably with synthetic organic chemistry pathways and in one example an 8 step chemical pathway was achieved in a three step enzyme pathway. |
Title | Pyridinium salts and uses thereof |
Description | The present invention relates to pyridinium salts, uses of pyridinium salts as antimicrobials and medicaments and processes for their production. Polysubstituted pyridinium salts are valuable pharmacophores found in many biologically active molecules. Their synthesis typically involves the use of multistep procedures or harsh reaction conditions. Here, we report water-based phosphate mediated reaction conditions that promote the condensation of arylacetaldehydes with amines to give 1,3,5-pyridinium salts. The reaction, carried out at pH 6, provides conditions suitable for the use of less stable aldehydes and amines in this Chichibabin pyridine condensation. The evaluation of selected 1,3,5-trisubstituted pyridinium salts highlighted that they can inhibit the growth of S. aureus in the low µg/mL range. The synthetic accessibility of these compounds and preliminary growth inhibition data may pave the way towards the discovery of new anti-bacterials based on the 1,3,5-trisubstituted pyridinium scaffold. |
IP Reference | GB1705863.7 |
Protection | Patent application published |
Year Protection Granted | 2017 |
Licensed | No |
Impact | A paper has been published |
Description | An invited talk entitled 'Green Chemistry: Enzymatic and Chemoenzymatic Strategies in Synthesis', at the 10th Choshu-London Symposium, Yamaguchi University, Japan, on 15th March 2018. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Part of the Tohoku Organic Chemistry Symposium, Tohoku University, Sendai, Japan, on 12th March 2018 |
Year(s) Of Engagement Activity | 2018 |
Description | An invited talk entitled 'Green Chemistry: Enzymatic and Chemoenzymatic Strategies in Synthesis', at the Tohoku Organic Chemistry Symposium, Tohoku University, Sendai, Japan, on 12th March 2018 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | A short lecture talk in Japan to enhance collaborations. |
Year(s) Of Engagement Activity | 2018 |
Description | Conference presentation entitled 'Biocatalytic Routes to Amines' at the Catalysis Hub |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | To highlight to the catalysis community how amines, including alkaloids, can be formed using biocatalysts |
Year(s) Of Engagement Activity | 2018 |
URL | https://www.rc-harwell.ac.uk/uk-catalysis-hub-summer-conference-2018 |
Description | Departmental seminar at Oxford entitled 'The use of enzymes for C-C bond formation and amine synthesis' |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | Departmental seminar to PDRAs, lecturers, postgraduates, undergraduates |
Year(s) Of Engagement Activity | 2018 |
Description | Departmental talk at the Technical University of Delft on 16th April 2018 entitled 'Biocatalytic routes to tetrahydroisoquinoline alkaloids' |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | To introduce how reaction cascades can be constructed to alkaloids |
Year(s) Of Engagement Activity | 2018 |
Description | Gordon Research Conference- Invited seminar |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Talk entitled 'Norcoclaurine Synthases and Enzymatic and Chemoenzymatic Cascades' |
Year(s) Of Engagement Activity | 2018 |
URL | https://www.grc.org/biocatalysis-conference/2018/ |
Description | Industrial Synthetic Biology Congress 2018 - invited talk entitled 'Synthesising Chemicals Using Synthetic Pathways In Plants' |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | Highlighting biological routes to making complex alkaloids |
Year(s) Of Engagement Activity | 2018 |
URL | https://www.oxfordglobal.co.uk/industrialsyntheticbiology-congress/ |
Description | Invited lecture at international conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Invited lecture on 'Enzymatic synthesis of novel halogenated alkaloids using a 'parallel cascade' strategy in vitro', at BIOTRANS I GRAZ 2021 on July 18th 2021 (on zoom). |
Year(s) Of Engagement Activity | 2021 |
Description | Invited speaker at Biocascades Symposium 2018, Bielefeld2018, entitled 'Enzyme cascades to tetrahydroisoquinolines using norcoclaurine synthases' on 10th April 2018 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | The conferences covered biocatalytic cascades with a mixture of industrial and academic speakers |
Year(s) Of Engagement Activity | 2018 |
Description | Inward mission on industrial biotechnology: 12 Feb 2018 |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Indian Industrial Biotech delegation at the UCL and will lead to some connections towards UK-India partnerships in future. Attendees · Dr. Annamma Anil Odaneth: Assistant Professor, Department of Biotechnology DBT- Institute of Chemical Technologies - Centre for Energy Biosciences, Institute of Chemical Technology, Mumbai, India: E: a.dbtceb@gmail.com · Dr Pramod Wangikar: Professor, Department of Chemical Engineering, Wadhwani Research Center for Bioengineering, Indian Institute of Technology Bombay Mumbai , India: E: wangikar@iitb.ac.in · Dr Rajkumar Rajagopal: Founder & Managing Director, Cellzyme Biotech India Pvt Ltd, Coimbatore, India: E: rajkumar77@gmail.com · Dr Syed Shams Yazdani: Group Leader, Microbial Engineering, Group Coordinator, DBT-ICGEB Centre for Advanced Bio-Energy Research, International Centre for Genetic Engineering and Biotechnology, ICGEB, New Delhi : E: ssyazdani@gmail.com |
Year(s) Of Engagement Activity | 2018 |
Description | Poster entitled 'NORCOCLAURINE SYNTHASE: A USEFUL TOOL WHICH ALLOWS ACCESS TO TETRAHYDROISOQUINOLINE ALKALOIDS', Gordon's conference, Biddeford, Maine, July 8 - 13, 2018 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | A poster entitled 'NORCOCLAURINE SYNTHASE: A USEFUL TOOL WHICH ALLOWS ACCESS TO TETRAHYDROISOQUINOLINE ALKALOIDS' at the Gordon's seminar, Biddeford, Maine, July 7-8, 2018, and at Gordon's conference, Biddeford, Maine, July 8 - 13, 2018. A number of discussions arose including potential collaborations and the poster was presented by Daniel Mendez-Sanchez |
Year(s) Of Engagement Activity | 2018 |
URL | https://www.grc.org/biocatalysis-conference/2018/ |
Description | Royal Society of Chemistry sponsored seminar at the Department of Biosciences, Exeter |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Undergraduate students |
Results and Impact | An invited RSC sponsored seminar entitled 'Biocatalytic Strategies to Isoquinoline Alkaloids' at the Department of Biosciences, Exeter. Discussion with undergraduates after the talk about using biocatalysts to make pharmaceuticals |
Year(s) Of Engagement Activity | 2017 |
Description | Talk at an international conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | A talk entitled 'Biocatalytic routes to tetraisoquinoline alkaloids using norcoclaurine synthases', at Biotrans2017, Budapest, Hungary |
Year(s) Of Engagement Activity | 2017 |
Description | University visit (Warwick) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Undergraduate students |
Results and Impact | Seminat at Warwick University to undergraduates and postgraduates entitled 'The use of enzymes for C-C bond formation and amine synthesis' describing the results arising from this project |
Year(s) Of Engagement Activity | 2017 |