ERA-IB5-The exploitation of Xanthophyllomyces dendrorhous as a sustainable platform for the PROduction of high-value CARotenoids

Lead Research Organisation: Royal Holloway University of London
Department Name: Biological Sciences

Abstract

Carotenoids pigments are used commercially as bioactives in cosmetics and health supplements, additives in a variety of feedstuffs, as fragrances, flavours and colorants as well as important industrial raw materials and fine chemicals. Their industrial relevance also means they are compounds of high-value with global markets in the range of $ 1 billion per annum. To date chemical synthesis has been the production method of choice. Such production remains expensive, creates reaction contaminants, non-native stereo isomers and is typically associated with chemical refineries using non-renewable energy sources. Numerous plant sources exist for carotenoids. However they are typically secondary metabolites often being produced in specialised cell types, organs or tissues at a specific developmental stage of the plant's life cycle by underutilised, slow growing and rare species that are not amenable to modern agricultural practices. Plant cell culture is highly prone to contamination and the size of the cells restricts biomass production in comparison to microbial sources. Algal sources require intense light, significant energy input during down-stream processing and the molecular resources are not adequately advanced to deliver competitive yields.
The onset of modern molecular approaches to pathway engineering and strain improvement, with the concurrent advances in fermentation technologies and down-stream processing means microbial production is a competitive sustainable alternative to plant and chemical based production. This approach also does not compete with land resources for food production or impact on our dwindling sources of fossil fuel. Therefore the development of microbial platforms provides a logical solution to the creation of renewable biosources of valuable chemicals with improved economic and environmental credentials. In the proposed project the red yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) will be developed as a microbial based renewable source of high-value carotenoids namely zeaxanthin and phytoene. Driven by demand the markets for these two carotenoids are rapidly growing. It is estimated that the global market for zeaxanthin will reach $334 million by 2018 (BCC Research report FOD025C). This interest in zeaxanthin is associated with its use as a treatment and preventative measure against Age Related Macular Degeneration (AMD). The chronic eye disorder AMD accounts for 54% of all cases of blindness in Western societies. Phytoene is a colourless carotene used as a bioactive ingredient in the rapidly expanding cosmetic industry (Von Oppen-Bezalel et al. 2007, SöFW-Journal 07). Its beneficial activities include protection against UV and oxidative damage, which lead to pre-mature ageing and other phenomena. As a production system for carotenoids Xanthophyllomyces has the advantage of being a naturally high producer of geranylgeranyl pyrophosphate (GGPP) derived terpenoids. For example, most bacteria and yeasts (such Saccharomyces) produce high levels of farnesyl diphospate (FPP), they do not synthesis longer chain GGPP derived terpenoids such as carotenoids. Thus intrinsic metabolism and regulatory mechanisms to synthesise and accumulate such compounds have evolved in this organism. Technological advances mean that Xanthophyllomyces now has a sequenced and annotated genome, established metabolomic platforms and is readily amenable to transformation with a suite of combinatory vectors capable of integrating multiple genes into the genome in a stable manner and can utilise agro-industry waste as carbon sources. Mutants accumulating different isoprenoid precursors are available and fermentation procedures from the laboratory to industrial scale established. Thus the project will exploit timely developments to create a microbial based platform capable of delivering renewable sources of high-value carotenoids.

Technical Summary

Most of the colours in the natural world are due to the presence of compounds known as carotenoids. These natural pigments have been utilised by human civilisations for millennia, and exploited by industry commercially as colorants, health and dietary supplements. Two particular carotenoids namely zeaxanthin and phytoene are in high demand and it is estimated that the global market for zeaxanthin only will reach $334 million by 2018. This interest in zeaxanthin is associated with its use as a treatment and preventative measure against Age Related Macular Degeneration (AMD), a chronic eye disorder that accounts for over half of all cases of blindness in Western societies. Phytoene is a colourless carotene used as a bioactive ingredient in the rapidly expanding cosmetic industry. Its beneficial activities include protection against UV and oxidative
damage, which lead to pre-mature ageing and other phenomena. Presently chemical synthesis is the production method used to produce these pigments/compounds. However, with the advent of new technologies naturally occurring microbial production is a competitive sustainable alternative to plant and chemical based production. This approach also does not compete with land resources
for food production or impact on our dwindling sources of fossil fuel. In the PROCAR project state of the art technologies will be used to develop the red yeast Xanthophyllomyces dendrorhous as a renewable source of natural zeaxanthin and phytoene to meet the demands of the European consumer.

Planned Impact

Not applicable-Pathways to Impact provided
 
Description The PROCAR project has been active since June 2015. Several findings have been obtained already.
1. Mutants have been obtained throughout the carotenoid pathway. These mutants exist as multiple alleles.
2. A metabolomics platform has been established for Phaffia that enables the detection of 120 compounds in one chromatographic system.
3. A zeaxanthin and phytoene prototype strains have been created.
4. The genes underlying most of the mutants in the cariotene pathway have been identified.
5. Scale up has been achieved.
6. Developed a CRISPR system for Phaffia
7. New gene targets not directly involved in the carotene pathway have been identified.
8. Elucidated mechanisms associated with the deposition of carotenes in fungi.
9. GC/MS libararies for non-conventional yeasts.
Exploitation Route 1. The metabolomics data is being used to create a genome scale model by a Danish collaborator.
2. A Horizon 2020 application was made but unsuccessful.
3. A horizon 2020 application is an stage two.
4. Responsive mode application to sequence mutants with Erlham.
5. Collaborative EU grant
6. ERA CoBio application.
Sectors Agriculture

Food and Drink

Chemicals

Education

Manufacturing

including Industrial Biotechology

Retail

URL http://www.disco-fp7.eu
 
Description Industrial partner is looking at capturing IPR on a range of mutants with altered and higher terpenoids. The range of mutants has been extended and sequencing will provide new targets for engineering. The mutants are being sequenced to identify new targets for isoprenoid biosynthesis enhancement. Three grant applications made to further the work and commericalise.
First Year Of Impact 2020
Sector Agriculture, Food and Drink,Chemicals,Education,Manufacturing, including Industrial Biotechology
Impact Types Societal

Economic

 
Description A synthetic metabolon for the production of valuable ketocarotenoids
Amount £50,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2016 
End 06/2017
 
Description College studentship
Amount £120,000 (GBP)
Funding ID College 
Organisation Royal Holloway, University of London 
Sector Academic/University
Country United Kingdom
Start 08/2016 
End 09/2019
 
Description college funding scheme
Amount £14,000 (GBP)
Organisation Royal Holloway, University of London 
Sector Academic/University
Country United Kingdom
Start 08/2018 
End 09/2019
 
Title MS library 
Description The MS GC libraries for compound and species identification 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact The characterisation of industrial strains by chemotyping 
URL http://www.rhul.ac.uk
 
Title Compound libraries 
Description GC-MS metabolite profiling compound libraries 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Chemotyping indutrial strains 
URL http://www.rhul.a.cuk
 
Description DTU 
Organisation Technical University of Denmark
Country Denmark 
Sector Academic/University 
PI Contribution Data to generate genome scale model
Collaborator Contribution Data to generate genome scale model
Impact in progress
Start Year 2015
 
Description MRM health 
Organisation MRM Health NV
Country Belgium 
Sector Private 
PI Contribution Apocarotenoid patent
Collaborator Contribution microbiology
Impact multi-disciplinary
Start Year 2021
 
Description Oxford Biotrans 
Organisation Oxford Biotrans Ltd.
Country United Kingdom 
Sector Private 
PI Contribution Supply of feedstocks with high beta carotene
Collaborator Contribution Access to explore the use of the P450 libarary
Impact Grant application to the BBSRC high value chemical production call
Start Year 2019
 
Description Wacker 
Organisation Wacker Chemicals
Country Global 
Sector Private 
PI Contribution market evalaution
Collaborator Contribution market evalaution
Impact in progress
Start Year 2015
 
Description metabolite analysis 
Organisation Syngenta International AG
Department Syngenta Ltd (Bracknell)
Country United Kingdom 
Sector Private 
PI Contribution Staff time to perform the analysis of volatiles
Collaborator Contribution Acess to machine time and germplasm resources
Impact The project still has one year to run -so to early
Start Year 2019
 
Description proplanta(ROmania 
Organisation PROPLANTA
Country Romania 
Sector Private 
PI Contribution Large scale extractions of zeaxanthin
Collaborator Contribution Equipment and resources
Impact Publications on the way and other grants
Start Year 2016
 
Title Carotenoid standards 
Description Carotenoid reference standards 
Type Of Technology New Material/Compound 
Year Produced 2022 
Impact RHUL are selling the carotenoid reference standards. 
 
Description College Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Providd explaination of the project and demnstrated the analytical equipment
Year(s) Of Engagement Activity 2019
 
Description College Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Open days involves presentations to the general public and prospective students - as well as demonstarting the analytical platforms
Year(s) Of Engagement Activity 2019
 
Description College Open days (Egham) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Open daya to promote the department and relay the research in the department
Year(s) Of Engagement Activity 2016,2017
 
Description College open days 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact College open days talks on the projects and discussion groups.
Year(s) Of Engagement Activity 2022
 
Description College public Open days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Routine 50 school pupils or general public are shown theresearch facilities and ongoing projects are described. A Q and A session is included.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016
URL https://www.royalholloway.ac.uk/science/sciencefestival/home.aspx
 
Description General public 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Communication forum on New plant breeding techniques "People Jury" virtual
Year(s) Of Engagement Activity 2021
 
Description Interview 
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 Article for popular magazine in international journal
Year(s) Of Engagement Activity 2018
 
Description M3 innovation 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Supporters
Results and Impact Engagement with local industry in the region
Year(s) Of Engagement Activity 2018
 
Description New genetic technologies for substainable 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 20 students Bracknell and Wokingham college A-level and BTEC students practical and talk.
Year(s) Of Engagement Activity 2024
 
Description School visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk and forum by Dr Enfissi on healthy diets and plant based foods
Year(s) Of Engagement Activity 2021
 
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 Dr Enfissi provide talks and practical classes (separation of colours by chromatography) to school children.
Year(s) Of Engagement Activity 2019
 
Description School visit (Bracknell) 
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 "60 school pupils (aged 11) attended for a school visit to the research organisation, which sparked questions and discussion afterwards, and the school reported increased interest in related subject areas.
Year(s) Of Engagement Activity 2017,2018
 
Description Schools taster day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact School science taster day
Year(s) Of Engagement Activity 2020
 
Description Verona (Italy) 
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 Training schools organised in the UK
Year(s) Of Engagement Activity 2017