13TSB_SynBio: Feasibility study on application of synthetic biology approaches to high yield production of Mycosporine-like Amino Acids for use as ...
Lead Research Organisation:
Newcastle University
Department Name: Inst for Cell and Molecular Biosciences
Abstract
Solar radiation reaching the Earth consists of infrared (>800 nm), visible (PAR, 400-750 nm) and ultraviolet-A (UVA, 320-400 nm). Damage to skin through exposure to ultra violet (UV) radiation is a major societal concern and consequently there is a significant consumer demand for cosmetics formulated to block UV penetration. Materials used to block UV penetration to the skin include inorganic metal oxide particles and organic filters. Currently, organic UV-absorbing compounds are manufactured from non-renewable petrochemicals and, as a result, there is considerable commercial interest in developing organic UV-absorber compounds from renewable materials. Croda wish to develop a sustainable manufacturing process for efficient, high-level production of a natural occurring group of UV-absorbing compounds, namely Mycosporine-like amino acids (MAAs), for use as a sunscreen additive to skin care products. Currently, the commercial production of MAAs for use in sunscreens is not a viable proposition due to the low yield and lack of fermentation technology associated with native MAA-producing organisms. This project will overcome this limitation by expressing genes encoding known MAA biosynthetic pathway enzymes in a bacterium that is widely used in industry to produce house-hold and food products and that has an excellent track record for safety.
Technical Summary
not required on previous TSB approved application.
Planned Impact
Who will benefit from this research and how will they benefit? The project will benefit both Croda and Newcastle University through increased integration and collaboration across a key strategic area of activity, namely synthetic biology. The application of this technology to a defined commercially driven application will benefit academic staff through generating truly translational research. This project has the potential to provide Croda with platform technologies to exploit for the sustainable production novel metabolites or metabolites currently produced from oil-based technology.
It is expected that the inter-disciplinary nature of the project will provide a platform for new career opportunities for the appointed Research Assistants and hence the project will become a source of highly trained researchers with significant added cross-disciplinary skills (i.e. between microbiology, bioinformatics, biomedical sciences, biochemistry, fermentation and downstream processing).
What will be done to ensure benefits result from the Project? The results arising from the project will be used by Croda to develop a sustainable industrial process with significant commercial potential. The resulting platform technologies will also provide Croda with sustainable approaches for the development of new products or the replacement of existing products manufactured from oil-based technologies.
It is expected that the inter-disciplinary nature of the project will provide a platform for new career opportunities for the appointed Research Assistants and hence the project will become a source of highly trained researchers with significant added cross-disciplinary skills (i.e. between microbiology, bioinformatics, biomedical sciences, biochemistry, fermentation and downstream processing).
What will be done to ensure benefits result from the Project? The results arising from the project will be used by Croda to develop a sustainable industrial process with significant commercial potential. The resulting platform technologies will also provide Croda with sustainable approaches for the development of new products or the replacement of existing products manufactured from oil-based technologies.
Publications
Borriss R
(2018)
Bacillus subtilis, the model Gram-positive bacterium: 20 years of annotation refinement.
in Microbial biotechnology
Errington J
(2017)
Designer chemistry.
in Environmental microbiology reports
Sauer C
(2016)
Effect of Genome Position on Heterologous Gene Expression in Bacillus subtilis: An Unbiased Analysis.
in ACS synthetic biology
Sauer C
(2018)
Exploring the Nonconserved Sequence Space of Synthetic Expression Modules in Bacillus subtilis.
in ACS synthetic biology
Glenwright H
(2016)
The Identification of Intrinsic Chloramphenicol and Tetracycline Resistance Genes in Members of the Bacillus cereus Group (sensu lato).
in Frontiers in microbiology
| Description | We took genes for synthesis of a natural sun tan protection molecule from a photosynthetic bacterium and engineered them for expression in an industrial bacterium, Bacillus subtilis. The results were partly successful in that the cells made a partial derivative of the molecule we wanted, but our industrial partner, Croda plc, are interested in following up the work to see if this molecule has commercialisable properties. |
| Exploitation Route | We are in discussions with Croda about possible follow on funding opportunities. |
| Sectors | Chemicals,Manufacturing, including Industrial Biotechology |
| Description | We are planning follow up proposals with Croda. |
| First Year Of Impact | 2016 |
| Sector | Manufacturing, including Industrial Biotechology |
| Title | B. subtile integration vector encoding a refactored Shinorine metabolic pathway cluster |
| Description | The tool comprised an integration vector with an inducible Shinorine gene cluster from Anabena refactored for expression in B. subtilis and strains in which the cluster has been integrated |
| Type Of Material | Cell line |
| Year Produced | 2015 |
| Provided To Others? | Yes |
| Impact | The impact was limited because of the failure to secure additional funding. A DTP PhD studentship was awarded to the project as a CASE award with Croda, but the student pulled out at the last minute and the studentship was awarded to another project. |
| Title | B subtilis metabolic model |
| Description | In collaboration with Dr Anil Wipat a previously developed metabolic model was extended to model flux associated with MAA synthesis |
| Type Of Material | Computer model/algorithm |
| Provided To Others? | No |
| Impact | The model was used to identify the likely route in B subtilis for MAA synthesis and to design the growth regime to maximise product formation. |
| Description | Application of Synthetic Biology Approaches fo the production of natural sunscreens |
| Organisation | Croda International |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | Metabolic engineering of Bacillus subtilis for the production of mycosporine like amino acids |
| Collaborator Contribution | fermentation of strains, extraction and analysis of metabolites |
| Impact | Engineered strains provided to Croda who have evaluated them for potential applications. Although a UV absorbing compound was detected in the engineered strain, the yield was insufficient to warrant further commercial development. However, the project provide proof of principle for the use of a Synthetic Biology approach to the production of novel metabolites |
| Start Year | 2013 |
| Description | Croda Europe Ltd, production of mycosporine-like amino acid for use as sunscreens |
| Organisation | Croda International |
| Department | Croda Chemicals Europe Ltd |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | Mycosporine-like amino acids (MAAs) are produced by some microbes in response to UV-irradiation. They have significant potential as additives to protect against UV-induced skin damage. The limitation to commercialisation is low yield of MAAs in the native producing organism. This project aimed seek to overcome this limitation by cloning the biosynthetic pathway into a suitable expression host. The ability of the host to produce high levels of the principal precursor metabolites is a key factor and required an evaluation of carbon flux through core metabolic pathways. The project was divided into a number of work packages with responsibility divided between the partners under overall management by Croda. The Newcastle Group was responsible for: WP1: Biosynthetic pathway manipulation. MAA synthesis draws on components of core metabolism from the pentose phosphate and amino acid synthetic pathways. Strategies to optimise flux to MAA synthesis were developed using metabolic modelling in silico. The model was sufficiently robust to allow recommendations to be made concerning the growth regime that should maximise product formation. Host strains were ultimately not modified to increase flux through the MAA pathway for reasons outlined below. WP2: Synthetic pathway cloning into B.subtilis host the gene cluster encoding the metabolic pathway for shinerine from Anabaena variables. The gene cluster was refactored and synthesised in vitro following codon optimisation and the selection of appropriate promoter, RBS and regulatory sequences, using Synthetic Biology principle and standards. Th host wasB. subtilis, which has previously been widely used for the large-scale industrial production of metabolites. The gene cluster was successfully synthesised and cloned into an integration vector for expression in B. subtilis. North blot analysis confirmed a good level of expression of all four genes in the cluster, conditional on the addition of the inducer. However, the strains showed little or no increased resistance to UV light, as might have been expected. As a result, a proteomics analysis was carried using 2D-PAGE and this revealed that on two of the proteins encoded by the cluster were visible in the cytoplasm of the cells. It was concluded that these proteins, either through lack of an associated folding pathway, or due to innate protease sensitivity, were subject to rapid intracellular degradation. Attempts were made to use UV light resistance to find suppressor mutants were unsuccessful. This feasibility study clearly demonstrated the limitations of Synthetic Biology in that while standardised parts and protocols can be used to generated strains, it cannot yet take into account the fact that native chassis strains such as E. coli and B. subtilis are complex organisms that have evolved sensitive and highly effective regulatory mechanism for survival in their native environments and that sometimes these regulator mechanisms (often referred to as quality control mechanisms) can work against even the most sophisticated engineered designs. |
| Collaborator Contribution | Croda Europe are responsible for production process, including downstream product recovery. The final product will be fully characterised for chemical identity, UV-absorption profile, and formulability in typical sunscreens. It is anticipated that this will present a platform capability to drive future development of alternate forms with enhanced characteristics. WP3: Production of MAA. Clones from two B. subtilis backgrounds were analysed by Croda for MAA production and they were able to detect new UV absorbing compounds in one of the clones. This was probably the result of the fact that the detected enzymes were the first two in the MAA pathway. Cord therefore evaluate influence of fermentation parameters on productivity and recovery of MAA. WP4 Utility of MAA in sunscreens. The MAA product will be characterised for chemical identity and purity.has been assess for formulability in typical sunscreen formulations. However, as far as we have been made aware, the detected product is unlikely to be developed commercially WP6: Project Management. The project was by managed by Croda. |
| Impact | A potential strain of B. subtilis was generated that produced a novel MAA that was evaluated by Croda for commercial exploitation, but not taken further As a feasibility project it met most of its criteria - attempts to get a PhD student to continue the work looked to be successful but the student pulled out at the last minute and the studentship was reallocated to another project by the DTP. |
| Start Year | 2013 |
| Description | Modelling mycosporine-like amino acid synthesis in Bacillus subtilis |
| Organisation | Newcastle University |
| Department | School of Computing Science |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Professor Anil Wipat supervised the development of the B. subtilis metabolic model |
| Collaborator Contribution | An existing data model was adapted to model the impact of mycosporine-like amino acid synthesis in Bacillus subtilis on flux through carbon and nitrogen pathways and to proposes optimal growth conditions |
| Impact | This is a multidisciplinary collaboration in which input from the model came in part from my involvement in the EU-funded BaSysBio project and Prof Wipat's expertise in modelling and bioinformatics |
| Description | "Why bacteria could be the answer to a future without oil." Article in The Conversation (Jan 5th 2015) |
| Form Of Engagement Activity | A magazine, newsletter or online publication |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Public/other audiences |
| Results and Impact | Wrote an article for the Conversation about how synthetic biology will solve many of the problems associated with leaving the oil-based economy behind. The article had massive impact wit (to date): 73,605 readers; 73 tweets; and 96 shares. |
| Year(s) Of Engagement Activity | 2015 |
| URL | https://theconversation.com/why-bacteria-could-be-the-answer-to-a-future-without-oil-35443 |
| Description | Advances in Microbiology and Biotechnology. Lviv |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Special guest and invited speaker in this meeting aimed at the biotechnology of microorganisms. I presented a talk entitled "Bacillus subtilis as the model gram- positive industrial bacterium" |
| Year(s) Of Engagement Activity | 2018 |
| URL | http://www.cellbiol.lviv.ua/2018/ |
| Description | Bacterial Protein Export Conference, Leuven |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | An invited speaker at this research meeting that brought together a high proportion of the world's leading experts on bacterial protein secretion to report on their current research. BPE2018 will provide an overview of recent exciting advances in the study of the various bacterial protein transport pathways, ranging from fundamentals to their impact on pathogenesis and biotechnology and the development of novel anti-bacterials. We will discuss the underlying molecular mechanisms of all known and emerging protein translocation modes, the structure and assembly of secretion channels, the biogenesis of integral membrane proteins, the structural dynamics of exported proteins and their interaction with chaperones, the use of signals for targeting and specific sub-cellular localization, the regulation and energetics of protein transport. The conference will cover from fundamental bacterial genetics and biochemistry to structural biology, protein dynamics and advanced single molecule biophysics. |
| Year(s) Of Engagement Activity | 2018 |
| URL | https://rega.kuleuven.be/bac/economou/bacterial-protein-export |
| Description | CBMNet meeting Factories for advanced biomanufacturing, Sheffield |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | The imperative to revolutionise the chemicals industry by creating a sustainable bio-based future and the increasing importance of biologics in medicine pose major challenges to UK biotechnologists. The design and implementation of bespoke advanced microbial cell factories, that can reproducibly yield bio-based alternatives to the chemicals that underpin so much of modern infrastructure is a fundamental challenge. Chassis engineering represents the single most critical technology to revolutionise biomanufacturing by improving product yields, simplifying product recovery and improving sustainability through reduced materials use and waste, thereby enhancing process economics and commercial viability. With a strong research base already working on microbial chassis engineering, CBMNet is the natural progenitor for such an event centred on microbial chassis design. |
| Year(s) Of Engagement Activity | 2017 |
| URL | https://cbmnetnibb.group.shef.ac.uk/members-forum/event-reports/december-2017-factories-for-advanced... |
| Description | Cesar Meeting on Antibiotic Resistance Sveti Martin |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | This meeting is part of the very active Croatian Microbiology Society. I gave a talk relating to the issue of non-expressed antibiotic resistance genes in Bacillus species and the regulatory implications in relation to release into the environment. |
| Year(s) Of Engagement Activity | 2018 |
| URL | https://fems-microbiology.org/opportunities/central-european-symposium-antimicrobials-antimicrobial-... |
| Description | European Bacillus meeting and Symposium on Central Carbon Metabolism, Paris |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | This is a long-standing series of meeting that I helped to establish in the 1990s and are still organised on an annual basis. I organised the meeting in Newcastle in 2015. The mission of the meeting is to bring together researchers from the European Bacillus community as well as members from several European consortia that perform fundamental and applied research on Bacillus subtilis and related Gram-positive bacteria. |
| Year(s) Of Engagement Activity | 2016 |
| URL | https://symposium.inra.fr/bacell2016/ |
| Description | European Bacillus meeting, Bath |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | This was the latest meeting of the European Bacillus community (BACELL), that I helped to establish in 1990s. It particularly provided young researchers the opportunity to discuss their research work |
| Year(s) Of Engagement Activity | 2018 |
| URL | https://www.facebook.com/events/2017588131854888/ |
| Description | Meeting of FEFANA (EU Association of Specialty Feed Ingredients and their Mixtures) concerning draft revised regulations from the European Food Standards Authority (EFSA) |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Policymakers/politicians |
| Results and Impact | The aim of this meeting was to provide a detailed response to the EFSA document entitled "Guidance on the characterisation of microorganisms 1 used as feed additives or as production organisms". My remit was to lead the response to EFSA in relation to antibiotic resistance genes. |
| Year(s) Of Engagement Activity | 2017 |
| URL | https://www.wired-gov.net/wg/news.nsf/articles/Guidance+on+characterisation+of+microorganisms+used+a... |
| Description | SynGen - Genomics and Synthetic Biology Conference, London |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Industry/Business |
| Results and Impact | The SynGen Series UK, with over 600 senior-level delegates representing internationally renowned research & academic institutions, clinical research institutions and pharmaceutical companies. Over 20 case studies and presentations demonstrating the latest synthetic biology tools and their therapeutic applications, including 2 interactive streams: Synthetic Biology - Tool Development Synthetic Biology - Applications Co-located with the highly established Annual Next Generation Sequencing and Clinical Diagnostics Congress, Annual Single Cell Analysis Congress and the Annual Genome Editing Congress. Invited talk on The Bacillus Cell Factory: Recent advances in novel tool development and session chair |
| Year(s) Of Engagement Activity | 2018 |
| URL | https://www.oxfordglobal.co.uk/syntheticbiology-congress/ |