Understanding carbazole biosynthetic enzymes: potential for a versatile assay of acyl CoAs
Lead Research Organisation:
University of Aberdeen
Department Name: Chemistry
Abstract
Fatty acid coenzyme As (acyl-CoAs) ubiquitously exist in living organisms and play central roles in many biochemical reactions. Abnormal accumulation of acyl-CoAs in cells are often the diagnostic symptoms for many diseases (i.e. human metabolic diseases including diabetes, fatty acid oxidation deficiency (FAOD), and cancers). Considerable efforts have gone into developing potential diagnostic tools to measure acyl CoAs so far and yet the outcomes are far from ideal.
Acyl CoAs are also the key precursors for microbial fermentation of fatty acid-derived chemicals. Currently many of these chemicals are prepared from fossil-derived feedstocks which require energy intensive processing, and are considered by many as non-sustainable for the future. In this respect, microbial fermentation is an attractive alternative to produce these commodity chemicals and enhanced acyl CoA production is associated with increased production of fatty acid derived chemicals in engineered microbes. In making acyl CoA enhanced, a key challenge is often to quantify the levels of acyl CoA among engineered microbes but researchers have yet to find a reliable way of doing this.
In this study, we are going to investigate an enzyme-based system that has the ability to convert acyl-CoAs into coloured aromatic compounds under mild conditions. These aromatic compounds are common motifs in dye materials. Consequently, the incorporation of our enzymatic system into biological samples will offer a versatile and convenient assay to measure acyl CoA concentration within the given samples. The use of bacterial enzymes to accomplish chemical tasks is well established in the food industry. The benefits in terms of sustainable manufacturing are well documented. However, the application of enzymatic processes/industrial biotechnology processes are still underdeveloped in the fine chemical, pharmaceutical, agricultural and bioscience industries. By working out the catalytic capabilities of the two novel enzymes involved in transforming acyl CoAs into coloured aromatic compounds, we will gain the ability to develop their biotechnological potential to be used as a new sensitive assay that will enable us to directly measure acyl CoAs in various biological samples.
Acyl CoAs are also the key precursors for microbial fermentation of fatty acid-derived chemicals. Currently many of these chemicals are prepared from fossil-derived feedstocks which require energy intensive processing, and are considered by many as non-sustainable for the future. In this respect, microbial fermentation is an attractive alternative to produce these commodity chemicals and enhanced acyl CoA production is associated with increased production of fatty acid derived chemicals in engineered microbes. In making acyl CoA enhanced, a key challenge is often to quantify the levels of acyl CoA among engineered microbes but researchers have yet to find a reliable way of doing this.
In this study, we are going to investigate an enzyme-based system that has the ability to convert acyl-CoAs into coloured aromatic compounds under mild conditions. These aromatic compounds are common motifs in dye materials. Consequently, the incorporation of our enzymatic system into biological samples will offer a versatile and convenient assay to measure acyl CoA concentration within the given samples. The use of bacterial enzymes to accomplish chemical tasks is well established in the food industry. The benefits in terms of sustainable manufacturing are well documented. However, the application of enzymatic processes/industrial biotechnology processes are still underdeveloped in the fine chemical, pharmaceutical, agricultural and bioscience industries. By working out the catalytic capabilities of the two novel enzymes involved in transforming acyl CoAs into coloured aromatic compounds, we will gain the ability to develop their biotechnological potential to be used as a new sensitive assay that will enable us to directly measure acyl CoAs in various biological samples.
Technical Summary
We propose to investigate the capabilities of two novel enzymes, a new III ketosynthase (KSIII) (NzsJ) and a new aromatase (NzsI), from Streptomyces sp. MA37, involved in the formation of a carbazole alkaloid (CA). These heterotricyclic molecules are critical components of many biologically important and industrially relevant molecules (i.e. dye materials). We have made significant progress in understanding the chemical mechanisms of two key enzymes (NzsE&H) involved in the biosynthesis of this CA, the first step towards harnessing the biotransformation potential of this group of molecules. From our preliminary results, we have begun to understand the mechanisms of the new KSIII NzsJ and our data indicated the formation of an indole-fused hexanone intermediate. We intend to continue this work and elucidate the mechanism of this enzyme in detail through site-directed mutagenesis studies to locate its active site. We will also probe the capacity of this enzyme using modified substrates with structural diversity. We have also started to understand the mechanism of the novel aromatase NzsI using stable isotopic labelling studies. Our data indicates the formation of the aromatic heterotricyclic ring via a one-pot biotransformation. We will advance our initial mechanistic work by using substrates substituted with various functional groups. Substrate tolerance is a key feature for a successful industrial biocatalyst. We will synthesize acyl thioesters equipped with different chain length and functionalities (i.e. saturated and unsaturated ring systems) and probe the substrate flexibility of NzsJ and NzsI. Furthermore, we will identify new homologues of NzsJ and NzsI that possess enhanced kinetic profiles and broader substrate tolerance through genome mining and synthetic gene expression.
This will not only give us control on the biochemical transformations but also allow us to develop a versatile and efficient colorimetric assay for acylCoA in various biological samples.
This will not only give us control on the biochemical transformations but also allow us to develop a versatile and efficient colorimetric assay for acylCoA in various biological samples.
Planned Impact
Impact will be delivered by fulfilling the research programme's key objective which are to provide stakeholders in various sectors with our new technology.
The project will deliver impact across four main areas.
People: The program will deliver multidisciplinary training in the chemical and biological sciences to one PDRA. The UK has identified synthetic biology, industrial biotechnology and new technology to biosciences as key deficits in scientists' training for the future workforce and this program will address those needs. The project is at the cutting edge of industrial biotechnology and will provide excellent training for the PDRA to ensure the PDRA can develop a career in industry or academia. The PDRA will also have access to a variety of staff training courses run by the University of Aberdeen, which are designed to enhance a wide variety of transferable and career based skills. All of the aforementioned points will ensure that the recruited PDRA has enhanced job prospects upon completion of the program and this will help to enhance the UK's scientific skill base. The PI's (Deng) lab regularly hosts undergraduates from the UK or Europe as summer placement students. Both Deng and Trembleau (departmental recruitment officer) are very active in outreach programmes that engage with secondary school pupils. If funded we will offer summer placements (1-2 per year) to local pupils to experience research at the chemical biology interface. We also intend to create a range of practical biochemical experiments suitable for University undergraduates in the first instance at Aberdeen but that will be made open-access via the PI's webpage.
Education: Interaction with local secondary schools will also help to enthuse the next generation of scientists. The Co-I (Trembleau) has prior experience, in this area, having led various projects as part of recruitment programmes held in the Chemistry Department (i.e. spectroscopy in suitcase, School visits, British Science Week). Students from secondary schools in the North-East of Scotland, accompanied by their teachers, spend a full week undertaking research projects.
Society: The work will offer new technologies to the biosciences using sustainable manufacturing methods (i.e. biocatalysts). The success of the programme will offer diagnostic tools that can be used in screening campaigns for diseases. The early detection of these diseases has the potential to provide a proper evaluation and management in a timely fashion both in the National Health Service (NHS), and, more directly in everyday life. The tools developed in this programme can be also used as an indicator to improve microbial fermentation of commodity chemicals in the fine chemicals. Delivering in these areas will in turn have a direct and potentially considerable impact on the health of the UK population and sustainable future.
Economic: The generation of novel IP will help to develop new markets for commercial exploitation which will help the UK PLC to remain internationally competitive in the areas of chemical-biology, biosciences and industrial biotechnology. As part of the work program we will utilise existing links and aim to establish new collaborations with industry to maximise the commercial exploitation of the research. We would seek to transfer technology through service agreements or by technology licensing. We will consider founding our own spin out company as the project develops.
Knowledge: At a fundamental level the programme will deliver exciting new insights into new enzymes (biocatalysts), NzsJ and NzsI, involved in the biosynthesis of carbazole alkaloids and specifically their mode of action. This new knowledge will be of benefit to researchers both in academia and in industries who are engaged in developing new biotransformation technology to biosciences.
The project will deliver impact across four main areas.
People: The program will deliver multidisciplinary training in the chemical and biological sciences to one PDRA. The UK has identified synthetic biology, industrial biotechnology and new technology to biosciences as key deficits in scientists' training for the future workforce and this program will address those needs. The project is at the cutting edge of industrial biotechnology and will provide excellent training for the PDRA to ensure the PDRA can develop a career in industry or academia. The PDRA will also have access to a variety of staff training courses run by the University of Aberdeen, which are designed to enhance a wide variety of transferable and career based skills. All of the aforementioned points will ensure that the recruited PDRA has enhanced job prospects upon completion of the program and this will help to enhance the UK's scientific skill base. The PI's (Deng) lab regularly hosts undergraduates from the UK or Europe as summer placement students. Both Deng and Trembleau (departmental recruitment officer) are very active in outreach programmes that engage with secondary school pupils. If funded we will offer summer placements (1-2 per year) to local pupils to experience research at the chemical biology interface. We also intend to create a range of practical biochemical experiments suitable for University undergraduates in the first instance at Aberdeen but that will be made open-access via the PI's webpage.
Education: Interaction with local secondary schools will also help to enthuse the next generation of scientists. The Co-I (Trembleau) has prior experience, in this area, having led various projects as part of recruitment programmes held in the Chemistry Department (i.e. spectroscopy in suitcase, School visits, British Science Week). Students from secondary schools in the North-East of Scotland, accompanied by their teachers, spend a full week undertaking research projects.
Society: The work will offer new technologies to the biosciences using sustainable manufacturing methods (i.e. biocatalysts). The success of the programme will offer diagnostic tools that can be used in screening campaigns for diseases. The early detection of these diseases has the potential to provide a proper evaluation and management in a timely fashion both in the National Health Service (NHS), and, more directly in everyday life. The tools developed in this programme can be also used as an indicator to improve microbial fermentation of commodity chemicals in the fine chemicals. Delivering in these areas will in turn have a direct and potentially considerable impact on the health of the UK population and sustainable future.
Economic: The generation of novel IP will help to develop new markets for commercial exploitation which will help the UK PLC to remain internationally competitive in the areas of chemical-biology, biosciences and industrial biotechnology. As part of the work program we will utilise existing links and aim to establish new collaborations with industry to maximise the commercial exploitation of the research. We would seek to transfer technology through service agreements or by technology licensing. We will consider founding our own spin out company as the project develops.
Knowledge: At a fundamental level the programme will deliver exciting new insights into new enzymes (biocatalysts), NzsJ and NzsI, involved in the biosynthesis of carbazole alkaloids and specifically their mode of action. This new knowledge will be of benefit to researchers both in academia and in industries who are engaged in developing new biotransformation technology to biosciences.
Publications
Liu Y
(2019)
Enzymatic Reconstitution and Biosynthetic Investigation of the Bacterial Carbazole Neocarazostatin A.
in The Journal of organic chemistry
Wu L
(2020)
Fluorine biocatalysis
in Current Opinion in Chemical Biology
Wang S
(2021)
Peculiarities of promiscuous l-threonine transaldolases for enantioselective synthesis of ß-hydroxy-a-amino acids
in Applied Microbiology and Biotechnology
Maglangit F
(2021)
Bacterial pathogens: threat or treat (a review on bioactive natural products from bacterial pathogens).
in Natural product reports
Wang S
(2022)
A ribosomally synthesised and post-translationally modified peptide containing a ß-enamino acid and a macrocyclic motif.
in Nature communications
Valera A
(2022)
Characterization of a class II ketol-acid reductoisomerase from Mycobacterium tuberculosis.
in RSC advances
Wang S
(2022)
Aminoacyl chain translocation catalysed by a type II thioesterase domain in an unusual non-ribosomal peptide synthetase.
in Nature communications
Casolari F
(2023)
Exploring a Streptomyces wax synthase using acyl-SNACs as donor substrates.
in RSC chemical biology
Wang S
(2024)
Dehydroamino acid residues in bioactive natural products
in Natural Product Reports
Alrashdi S
(2024)
Chemo-Enzymatic Synthesis of Bioactive Carbazole Derivatives
in SynBio
Description | 1. Characterization of the key enzymes in the proposed programme. The paper has been published in Journal of Organic Chemistry 2020 (American Chemistry Society). 2. Overexpression key enzyme homologues identified from other organisms as proposed in the programme. 3. Characterization of key enzymes in the biosynthetic pathway of carbazole analogues identified in a streptomyces strain from Thailand and explore the catalytic capacity. 4. Biochemical analysis suggested that enzymes from Thai strain display relatively broad substrate specificity. manuscript is under preparation. |
Exploitation Route | Understanding of the catalytical capacity of these enzymes will help us to establish an efficient biotransformation system for future applications. Ingenza is a biotechnology company based on Edinburgh that is interested in this technology and supported the team in various ways, i.e. in-kind contribution. the success of the project may allow us to develop a commercial kit in medical environments. |
Sectors | Chemicals Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
Description | Developing a carbazole-base biosensor for the measurement of methacrylate intermediates |
Amount | £110,000 (GBP) |
Organisation | IBioIC |
Sector | Academic/University |
Country | United Kingdom |
Start | 09/2019 |
End | 08/2023 |
Description | Dissection of the cyclization mechanism underlying the assembly of the bacterial tricyclic carbazole natural products |
Amount | £12,000 (GBP) |
Funding ID | IEC\NSFC\170617 |
Organisation | The Royal Society |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2017 |
End | 04/2020 |
Title | A bioactive ribosomally synthesised peptide with unusual structural features |
Description | enzymes that make unusual unsaturated beta-enamide in a peptide and mediate a new cyclized moiety. |
Type Of Material | Biological samples |
Year Produced | 2022 |
Provided To Others? | Yes |
Impact | Will offer the bioengineering/synthetic biology community new tools to incorporate unsaturated beta-amino acid and new cyclic moieties into their corresponding peptide systems. |
URL | https://doi.org/10.1038/s41467-022-32774-3 |
Title | KASII enzymes and aromatases |
Description | identification and overexpression of a series of KASIII and aromatase homologues. currently the characterization is underway. |
Type Of Material | Biological samples |
Year Produced | 2018 |
Provided To Others? | No |
Impact | We will test these homologues of the KASIIIs and aromatases with the aim of finding the enzymes with the best kinetics and wider substrate specificity for the potential application in acyl-CoA measurement |
Description | Carbazole biosynthesis |
Organisation | Wuhan University |
Department | State Key Laboratory for Combinatorial Biosynthesis |
Country | China |
Sector | Academic/University |
PI Contribution | This is the long-term collaboration between me and the Wuhan partners. We focus on the in vitro characterization of the biosynthetic enzymes, intermediates and potential application of these enzymes for acyl-CoA measurement. |
Collaborator Contribution | The Wuhan partners focus on in vivo characterization of the biosynthetic pathways and isolation of intermediates, |
Impact | We have published several papers listed below in this project since 2015 prior the award. Preliminary data are gathered and manuscript about our recent progress is currently under the way. Org. Biomol. Chem., 2017, 15, 3843-3848. Org. Biomol. Chem., 2016, 14, 8679-8684. Chem. & Biol., 2015, 22(12), 1633-1642. it is a multi disciplinary involving chemistry, microbiology, enzymology and molecular biology. |
Start Year | 2017 |
Description | Lucite |
Organisation | Lucite International |
Department | Lucite International UK Ltd |
Country | United Kingdom |
Sector | Private |
PI Contribution | Lucite International is the leading company that manufacture methylcrylate chemicals. The R&D department has been interested in the above research and invited us to join its consortium of bio-based methylcrylate manufacturing. The student, PI and CoI participated the consortial meetings in 2019 and 2020. We have also had a 6-month review meeting since 2019. |
Collaborator Contribution | The partnership brought the expertise in the bio-based methylcrylate manufacturing. the industrial partners provided key suggestions/guidance related to industrial setting which are valuable for us to make further progress of the project. |
Impact | it is multi-displinary. we signed a confidential contract with Lucite to participate the consortial meetings. |
Start Year | 2019 |
Description | carbazale enzymes |
Organisation | Ingenza Ltd |
Country | United Kingdom |
Sector | Private |
PI Contribution | Ingenza is interested in the enzyme system that are developed by this award to solve potential issues in their biotransformation systems (confidential) and as such is keen to develop a partnership with the team. |
Collaborator Contribution | Ingenza supported HD's application to IBioIC CTP programme with in-kind cash contribution. The application was awarded and the student will start the collaborative programme later this year, 2019. |
Impact | The partnership resulted in a successful PhD application funded by IBioIC/University of Aberdeen. This is a multi disciplinary result, covering chemical synthesis, bioinformatics, enzymology, industrial biotechnology, synthetic biology and cell biology. |
Start Year | 2018 |
Description | Cell block science |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Discussed the team's research using hands on activities/games with prisoners at HMP Grampian and their families. |
Year(s) Of Engagement Activity | 2019 |
Description | One-day symposium with Chinese visitors and collaborators |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | The Chinese collaborators visited the research teams in Aberdeen, in July, 2020 under the funding support of NSFC-RS Newton Mobility scheme. The team hosted a symposium for the visitors and showcased the preliminary results and technologies from both awards. After the symposium, Chinese collaborators and the team decided to the next research activities and next scientific publications. |
Year(s) Of Engagement Activity | 2020 |
Description | Panel discussion |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Panel discussion with 4 scientists from University of Aberdeen led by the SHMU youth media team to a live audience of everyday people of all ages as part of the May Festival at University of Aberdeen. |
Year(s) Of Engagement Activity | 2019 |
Description | Radio host |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | host at SHMU radio to help the youth media team develop a science show of their own |
Year(s) Of Engagement Activity | 2018,2019,2020,2021 |
Description | Research visit to Chinese collaborators in Wuhan in 2019 |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Visit Chinese collaborators in the School of Pharmaceutical Sciences, Wuhan University and Renji Hospital, the School of Medical Sciences, Shanghai JiaoTong University, China, and discuss potential publication writings under the funding support of NSFC-RS newton mobility scheme, 13-19, April, 2019. |
Year(s) Of Engagement Activity | 2019 |
Description | School visit |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | St Joseph Primary School organised "Science Day" June, 2019 and invited HD to participate in this event. HD briefly introduced the principles of biochemistry and presented a colorimetric experiment including a series of enzymatic reactions to aspire school pupiles and general audience. The teachers told HD that it appeared that there have been increased interest in the school in biochemistry/chemistry. |
Year(s) Of Engagement Activity | 2019 |
Description | School visit |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Performed a "flashes and bangs" show for secondary school aged children focussing on the concepts of energy changes during chemical reactions. |
Year(s) Of Engagement Activity | 2019,2020 |
Description | committee member |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Industry/Business |
Results and Impact | Dr Kirstie Rickaby is the committee member of the SHINE (Scottish Health & Innovation Network for Entrepreneurship) committee . |
Year(s) Of Engagement Activity | 2020 |
Description | engagement training |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Supporters |
Results and Impact | 3 day course aimed at enhancing public engagement skills and at designing an event to be held in Oban during West Highland Yachting Week in July where various aspects of current ocean based research will be showcased. |
Year(s) Of Engagement Activity | 2018,2019 |
Description | outreach event |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | During 2 days of the West Highland Yachting Week in Oban, the events designed during the training programme were rolled out for the general public to try. |
Year(s) Of Engagement Activity | 2019 |
Description | radio show |
Form Of Engagement Activity | A broadcast e.g. TV/radio/film/podcast (other than news/press) |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Dr Rickaby, the appointed PDRA, is the presenter of a radio show called "Talking Science" that broadcasts live across Aberdeen every 2 weeks. Every episode covers the latest in science news and features an interview with 1 local researcher. The show emphasises communicating science to the development areas of the city and to people who may have less exposure to science in their everyday lives. |
Year(s) Of Engagement Activity | 2019 |