17-ERACoBioTech Thermophilic bacteria and archaeal chassis for extremolyte production -HotSolute
Lead Research Organisation:
UNIVERSITY OF EXETER
Department Name: Biosciences
Abstract
Thermophilic organisms are composed of both bacterial and archaeal species. The enzymes isolated from these species and from other extreme habitats are more robust to high temperature, organic solvents and to breakdown from other proteolytic enzymes. They often have unique substrate specificities and originate from novel metabolic pathways. Thermophiles as well as their stable enzymes ('thermozymes') are receiving increased attention for biotechnological applications.
The proposed project will establish thermophilic in vitro enzyme cascades as well as two new chassis, the thermophilic bacterium Thermus thermophilus (Tth) and the thermoacidophilic archaeon Sulfolobus acidocaldarius (Saci) as new thermophilic, bacterial and archaeal platforms for the production of novel high added-value products called 'extremolytes'.
Extremolytes are small molecule compatible solutes found naturally in the cells of thermophilic species that accumulate in the cell in response to multiple environmental stresses and help to stabilize cellular components (including proteins and membranes). Extremolytes offer an amazing so far unexploited potential for industrial applications including food, health, consumer care and cosmetics. However, their production in common mesophilic organisms such as fungi and Escherichia coli is currently hampered by the hyperthermophilic origin of the respective metabolic pathways which require a thermophilic cell factory.
The development of the newly designed 'cell factories' will be used for the production of three extremolytes, cyclic 2,3 di-phosphoglycerate (cDPG), di-myo-1,1'-inositol-phosphate (DIP) and mannosylglycerate (MG). These extremolytes (with few exceptions for MG) are exclusively found in hyperthermophiles, and have not been produced in a mesophilic host to date. The extremolyte biosynthetic pathways have been identified and many of the enzymes involved have been characterized. Within the project in addition to these well established enzymes, new candidates will be provided by searching for new synthetic enzymes in metagenome newly isolated strains from (hyper)thermophilic habitats.
All three extremolytes are derived in a few steps from central glycolytic intermediates and are absent in Saci and only MG has been reported in Tth. The establishment of thermophilic in vitro enzyme cascades as well as in vivo enzyme platforms will be used for extremolyte production. Both organisms, Saci and Tth are easy to grow (minimal or complex media, aerobic growth). Many other thermophilic organisms require anaerobic or specialised conditions to achieve successful growth in the laboratory or in an industrial setting.
Importantly advanced genetic tools have been established for both Tth and Saci that will allow the insertion of new modules into the cells using a synthetic biology approach. For enzyme cascade and strain design, construction, optimization and product recovery a model-based systems biology and synthetic biology approach will be employed including state of the art genetics, biochemistry, transcriptomics, proteomics, modelling, data management and life cycle assessment.
The proposed project will establish thermophilic in vitro enzyme cascades as well as two new chassis, the thermophilic bacterium Thermus thermophilus (Tth) and the thermoacidophilic archaeon Sulfolobus acidocaldarius (Saci) as new thermophilic, bacterial and archaeal platforms for the production of novel high added-value products called 'extremolytes'.
Extremolytes are small molecule compatible solutes found naturally in the cells of thermophilic species that accumulate in the cell in response to multiple environmental stresses and help to stabilize cellular components (including proteins and membranes). Extremolytes offer an amazing so far unexploited potential for industrial applications including food, health, consumer care and cosmetics. However, their production in common mesophilic organisms such as fungi and Escherichia coli is currently hampered by the hyperthermophilic origin of the respective metabolic pathways which require a thermophilic cell factory.
The development of the newly designed 'cell factories' will be used for the production of three extremolytes, cyclic 2,3 di-phosphoglycerate (cDPG), di-myo-1,1'-inositol-phosphate (DIP) and mannosylglycerate (MG). These extremolytes (with few exceptions for MG) are exclusively found in hyperthermophiles, and have not been produced in a mesophilic host to date. The extremolyte biosynthetic pathways have been identified and many of the enzymes involved have been characterized. Within the project in addition to these well established enzymes, new candidates will be provided by searching for new synthetic enzymes in metagenome newly isolated strains from (hyper)thermophilic habitats.
All three extremolytes are derived in a few steps from central glycolytic intermediates and are absent in Saci and only MG has been reported in Tth. The establishment of thermophilic in vitro enzyme cascades as well as in vivo enzyme platforms will be used for extremolyte production. Both organisms, Saci and Tth are easy to grow (minimal or complex media, aerobic growth). Many other thermophilic organisms require anaerobic or specialised conditions to achieve successful growth in the laboratory or in an industrial setting.
Importantly advanced genetic tools have been established for both Tth and Saci that will allow the insertion of new modules into the cells using a synthetic biology approach. For enzyme cascade and strain design, construction, optimization and product recovery a model-based systems biology and synthetic biology approach will be employed including state of the art genetics, biochemistry, transcriptomics, proteomics, modelling, data management and life cycle assessment.
Technical Summary
The proposed project will establish thermophilic in vitro enzyme cascades as well as two new chassis, the thermophilic bacterium Thermus thermophilus (Tth, 65-75degrees C, pH 7.0) and the thermoacidophilic archaeon Sulfolobus acidocaldarius (Saci, 75-80 degrees C, pH 2-4), as new thermophilic, bacterial and archaeal platforms for the production of novel high added-value products, extremolytes.
The development of the newly designed 'cell factories' will be used for the production of three extremolytes, cyclic 2,3 di-phosphoglycerate(cDPG), di-myo-1,1'-inositol-phosphate (DIP) and mannosylglycerate (MG). These extremolytes (with few exceptions for MG) are exclusively found in hyperthermophiles, and have not been produced in a mesophilic host to date. The project will use these existing natural biological cell based systems with a synthetic biology approach. New biological so called parts and devices will be employed in order to establish and optimize two new thermophilic 'cell factories' for the production of the novel extremolytes with medical and personal care applications.
Both organisms, Saci and Tth are easy to grow (minimal or complex media, aerobic growth). Importantly advanced genetic tools have been established for both Tth and Saci that will allow for the insertion of new modules using a synthetic biology approach. For enzyme cascade and strain design, construction, optimization and product recovery, a model-based systems biology and synthetic biology approach will be employed including state of the art genetics, biochemistry, transcriptomics, proteomics, modelling, data management and life cycle assessment.
During the project a bioinformatic approach will also be used to search for new enzymes from metagenomes and newly sequenced hyper-thermophilic genomes.
To provide small quantities of extremolytes to the industrial partners (SME and Evonik) for testing early in the project by isolation from the natural producer strain.
The development of the newly designed 'cell factories' will be used for the production of three extremolytes, cyclic 2,3 di-phosphoglycerate(cDPG), di-myo-1,1'-inositol-phosphate (DIP) and mannosylglycerate (MG). These extremolytes (with few exceptions for MG) are exclusively found in hyperthermophiles, and have not been produced in a mesophilic host to date. The project will use these existing natural biological cell based systems with a synthetic biology approach. New biological so called parts and devices will be employed in order to establish and optimize two new thermophilic 'cell factories' for the production of the novel extremolytes with medical and personal care applications.
Both organisms, Saci and Tth are easy to grow (minimal or complex media, aerobic growth). Importantly advanced genetic tools have been established for both Tth and Saci that will allow for the insertion of new modules using a synthetic biology approach. For enzyme cascade and strain design, construction, optimization and product recovery, a model-based systems biology and synthetic biology approach will be employed including state of the art genetics, biochemistry, transcriptomics, proteomics, modelling, data management and life cycle assessment.
During the project a bioinformatic approach will also be used to search for new enzymes from metagenomes and newly sequenced hyper-thermophilic genomes.
To provide small quantities of extremolytes to the industrial partners (SME and Evonik) for testing early in the project by isolation from the natural producer strain.
Planned Impact
The impact
This project will develop the application of thermophilic micro-organisms (both bacterial and archaeal) as cell factories for the
production of new industrially interesting molecules.
Thermophilic enzyme cascades will be developed in order to produce three high added-value products extremolytes, which have
applications in medical and skin care products.
The platforms once established can be used for production of other important products using a synthetic biology approach of enzyme
cascades for both in vivo and in vitro systems and drive new applications of industrial biotechnology.
Natural producer strains will be used to produce extremolytes for initial testing
The public visibility and dissemination events will make people increasingly aware of the importance and possibilities that research
and innovation in industrial biotechnology can offer for them. Increased knowledge in this area will be transferred from academia to industry
and will enable training of experienced personnel in this multidisciplinary area of sustainable bio-processing.
This project directly contributes to the aims of this ERA-CoBioTech call, to transform the global economy from a dependence on fossil raw materials to a sustainable bio-based economy. The development of this approach will enable future developments and innovations in response to societal and industrial needs within the bioeconomy.
In the longer term, it will further propagate the exploitation of the hot bacterial and archaeal chassis for the integration of other new synthetic modules that have direct applications of industrial biotechnology. The combination of new thermophilic features (e.g. pathways, enzymes and regulatory circuits) of bacterial, eukaroytic and archaeal origin offer new exciting possibilities for future applications in industrial biotechnology.
The production of the three new high-value compounds, extremolytes, cDPG, DIP and MG represents great innovative potential for the cosmetics and health care industries. The large scale production of extremolytes will trigger technological innovation by new patents for extremolyte employment and increase the established market for other extremolytes (e.g. ectoine, hydroxyectoine) in the cosmetic and
pharmaceutical industries.
These compounds are known to stabilize proteins, cell membranes and produce a lung surfactant monolayer. They are also used in anti-allergic creams as well as moisturizing nasal sprays, eye drops, inhalation solutions, derma products and as anti-aging and sun protection cosmetics. In this respect they will improve the overall health of the general public.
This project will develop the application of thermophilic micro-organisms (both bacterial and archaeal) as cell factories for the
production of new industrially interesting molecules.
Thermophilic enzyme cascades will be developed in order to produce three high added-value products extremolytes, which have
applications in medical and skin care products.
The platforms once established can be used for production of other important products using a synthetic biology approach of enzyme
cascades for both in vivo and in vitro systems and drive new applications of industrial biotechnology.
Natural producer strains will be used to produce extremolytes for initial testing
The public visibility and dissemination events will make people increasingly aware of the importance and possibilities that research
and innovation in industrial biotechnology can offer for them. Increased knowledge in this area will be transferred from academia to industry
and will enable training of experienced personnel in this multidisciplinary area of sustainable bio-processing.
This project directly contributes to the aims of this ERA-CoBioTech call, to transform the global economy from a dependence on fossil raw materials to a sustainable bio-based economy. The development of this approach will enable future developments and innovations in response to societal and industrial needs within the bioeconomy.
In the longer term, it will further propagate the exploitation of the hot bacterial and archaeal chassis for the integration of other new synthetic modules that have direct applications of industrial biotechnology. The combination of new thermophilic features (e.g. pathways, enzymes and regulatory circuits) of bacterial, eukaroytic and archaeal origin offer new exciting possibilities for future applications in industrial biotechnology.
The production of the three new high-value compounds, extremolytes, cDPG, DIP and MG represents great innovative potential for the cosmetics and health care industries. The large scale production of extremolytes will trigger technological innovation by new patents for extremolyte employment and increase the established market for other extremolytes (e.g. ectoine, hydroxyectoine) in the cosmetic and
pharmaceutical industries.
These compounds are known to stabilize proteins, cell membranes and produce a lung surfactant monolayer. They are also used in anti-allergic creams as well as moisturizing nasal sprays, eye drops, inhalation solutions, derma products and as anti-aging and sun protection cosmetics. In this respect they will improve the overall health of the general public.
Publications
Cutlan R
(2020)
Using enzyme cascades in biocatalysis: Highlight on transaminases and carboxylic acid reductases.
in Biochimica et biophysica acta. Proteins and proteomics
De Rose S
(2021)
Production of the Extremolyte Cyclic 2,3-Diphosphoglycerate Using Thermus thermophilus as a Whole-Cell Factory
in Frontiers in Catalysis
De Rose S
(2023)
Structural characterization of a novel cyclic 2,3-diphosphoglycerate synthetase involved in extremolyte production in the archaeon Methanothermus fervidus
in Frontiers in Microbiology
De Rose SA
(2021)
Biochemical and Structural Characterisation of a Novel D-Lyxose Isomerase From the Hyperthermophilic Archaeon Thermofilum sp.
in Frontiers in bioengineering and biotechnology
| Title | Postcard to advertise project to general public and other academics and industries |
| Description | Postcard for general advertising |
| Type Of Art | Image |
| Year Produced | 2019 |
| Impact | Allow circulation at different events and in public places |
| URL | https://twitter.com/hotsolute |
| Description | This research has already produced in vitro and in vivo expression of small molecule extremolytes in the thermophilic, bacterial host Thermus thermophilus in Exeter, UK. Our German partners are working with archaeal Sulfolobus acidocaldarus and can express extremolytes in vitro and have preliminary results of expression in vivo. It has previously not been possible to introduce these unique different synthetic pathways for expression of the extremolytes in microorganisms that grow at lower temperatures such as Escherichia coli. It is therefore necessary to express them in a thermophilic host such as Thermus thermophilus. Two unique enzymes, 2-phosphoglycerate kinase (2PGK) forming 2,3-di-phosphoglycerate and di-phosphoglycerate synthase (cDPGS), which introduces the cyclisation to the DPG have been biochemical and structural characterised since no known homologues are known at a sequence and structural level. Both enzymes have low sequence identity to other known enzymes available in the Protein Data Base. The cDPGS structure has been solved to high resolution using seleno-methionine due to no molecular replacement models being available. It has also been solved in complex with 2,3-diphosphoglycerate and ADP Mg2+ showing the conformational changes that occur on substrate binding . The structure appears unique with only one domain showing homology to other known proteins that have unrelated function. The structure of the other enzyme, 2PGK is currently being solved. The structures of the 2 novel enzymes within the extremolyte biosynthetic pathway will further the understanding of their catalytic mechanisms. The extremolytes have applications for the cosmetic and healthcare industries. A patent application using the cDPGS enzyme for large scale production is being taken forward by the partner company Evonik. |
| Exploitation Route | Exploitation for production of a range of different extremolytes will be taken forward initially by Evonik and later licensed to other companies. |
| Sectors | Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| URL | https://twitter.com/hotsolute |
| Description | The findings of this award are of special interest to the industrial partner Evonik, Germany and to an associated SME, Bitop, Germany The production of cDPG in a one step process is being investigated for patent application by our German partners. The extremolytes to be produced have both healthcare and medical applications and are high value compounds. We have been able to demonstrate the expression of the two unique enzymes that are required for the synthesis of cDPG and demonstrated the whole-cell production of this extremolyte in a thermophilic host Thermus thermophilus. This is not possible in the mesophilic host Escherichia coli. This demonstrates the possibility of using Thermus thermophilus for other in vivo synthesis of different extremolytes and related small molecules. This work has recently been published in the open access journal Frontiers Catalysis. |
| First Year Of Impact | 2021 |
| Sector | Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Societal,Economic |
| Description | ERA CoBioTech Strategic Agenda - a vision for biotechnology in Europe Foreword |
| Geographic Reach | Europe |
| Policy Influence Type | Participation in a guidance/advisory committee |
| Impact | Published document for policy makers |
| URL | https://www.cobiotech.eu/lw_resource/datapool/systemfiles/elements/files/7D5DE99D41EC4DCCE0539A695E8... |
| Description | ERA-CoBiotech |
| Amount | £412,012 (GBP) |
| Funding ID | BB/R02166X1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 03/2018 |
| End | 02/2021 |
| Title | Mass Spec data |
| Description | Mass Spec data to support publication in University of Exeter ORE |
| Type Of Material | Database/Collection of data |
| Year Produced | 2021 |
| Provided To Others? | Yes |
| Impact | Production and quantification of extremolyte production in Thermus thermophilus |
| Description | HotSolute ERA-CoBiotech |
| Organisation | University Duisburg-Essen |
| Country | Germany |
| Sector | Academic/University |
| PI Contribution | Using the thermophilic bacterium Thermus thermophilus as a thermophilic cell factory for the production of the extremolyte, Cyclic Diphosphoglycerate Characterisation of the enzymes involved both biochemically and structurally |
| Collaborator Contribution | Using the thermophilic archaeon Sulfolobus solfataricus as a thermophilic cell factory for production of several different extremolytes |
| Impact | https://www.frontiersin.org/articles/10.3389/fctls.2021.803416 Catal., 20 December 2021 | https://doi.org/10.3389/fctls.2021.803416 Production of the Extremolyte Cyclic 2,3-Diphosphoglycerate Using Thermus thermophilus as a Whole-Cell Factory Paper to be submitted 31st March 2023 to Frontiers in Microbiology - Structural characterisation of the novel cyclic 2,3-diphosphoglycerate synthetase from Methanothermus fervidus involved in extremolyte synthesis Simone A. De Rose, Michail N. Isupov, Harley L. Worthy, Nicholas J. Harmer, Jennifer A. Littlechild* and the HotSolute consortium Multidisciplinary collaboration University of Essen, Molecular biology of Sulfolobus solfataricus, metabolic pathways -publications in progress |
| Start Year | 2018 |
| Description | Hotsolute Grant. Collaborations with Evonik,Germany, Prof Siebers, University of Essen, Germany, Dr vDaniela Monti, ICRM,Milan, Italy, Prof Elizaveta Bonch-Osmolovskaya,Russia |
| Organisation | C.N.R.-Institute of Chemistry of Molecular Recognition |
| Country | Italy |
| Sector | Public |
| PI Contribution | We have been involved in the expression of enzymes for extremolyte production from different sources in the T. thermophilus system and detailed enzyme characterization for the establishment of reaction kinetic models. We have worked closely with P1 and to integrate the optimized modules for cDPG, and DIP in Tth for strain design in collaboration with P1, P3 and P5. A paper on extremolyte production of cyclic 2,3 di-phosphoglycerate in T. thermophilus has been achieved published. Characterisation and Structural studies on the unique cDPGS enzyme has been carried out and a paper is currently being submitted to Frontiers in Microbiology. Enzyme cascade and strain optimization will be performed with the aid of modelling by Exeter in collaboration with P1 and P6. Exeter has expertise in using the bacterial Tth system as a thermophilic cell factory and has knowledge with both commercially available expression plasmids for this organism and construction of new plasmids including a Biobrick Tth /E.coli vector together with promotor design, codon optimization . This expertise has been achieved partly as part of a BBSRC Case studentship with GSK as part of and enzyme cascade developed for biocatalysis. Tth has an added advantage of being naturally competent due to a specialized macromolecular DNA translocator assembly which allows the entry of foreign DNA past the cell envelope into the cell. We have also collaborated with the partner in Italy and partner in Russia with bioinformatics screening to look for new enzymes for extremolyte synthesis found in other thermophilic genomes and metagenomes with P5 including the DNA resource from the EU Hotzyme project where P1,P3,P5 collaborating together. This has involved the cloning of the thermophilic genes related to the cDPG and 2PGK genes in E.coli and protein characterization with P3 and work with P1 on assay development. A variant of the 2PGK2 is currently being screened for crystallisation of this enzyme which produced only small crystals not suitable for X-ray characterisation from the original Methanococcus source. . The Exeter group has extensive experience in cloning thermophilic bacterial and archaeal enzymes and their industrial applications as summarized in two recent reviews and structural characterization . We have worked with P3 on development of the in vitro cascades of thermophilic enzymes involved in synthesis of other selected extremolytes and will investigate other pathways which could produce related products of industrial and pharmaceutical interest. Exeter and P3 have been part of a COST action on Systems Biocatalysis which has now finished. |
| Collaborator Contribution | The aim of HotSolute was to provide new extremolytes, i.e. cyclic 2,3 di-phosphoglycerate (cDPG), di-myo-1,1'-inositol-phosphate (DIP) and mannosylglycerate (MG), that cannot be produced in current mesophilic hosts by the use of thermophilic in vitro enzyme cascades and new thermophilic expression chassis. The thermophilic bacterium Thermus thermophilus (Tth) and the thermoacidophilic Archaeon Sulfolobus acidocaldarius (Saci) have been used as platform organisms. Studies using Sulfolobus as a thermophilic host have been carried out by the co-ordinator and further joint papers are in preparation. HotSolute has been coordinated by Bettina Siebers (P1, University of Duisburg-Essen, UDE) who was responsible for project and who has presented findings at on-line conferences with ERA-IB co-ordinators. Overall management of the project (e.g. organization of regular meetings, for all external reporting), for scientific and quality control of the project (e.g. internal reports) general adherence to data guidelines (SEEK, FAIRDOM), data standardization (SOPs)) and communication and dissemination (e.g. homepage, flyers) has been carried pout by partner 1 will help from all partners. The coordinator was assisted by Evonik industries (P4) in communication and dissemination aspects and by the Science Support Centre (SSC) at the UDE (e.g. consortium agreement, intellectual property rights). Scientifically, P1 has been involved in the expression of enzymes for extremolyte production from different sources in Saci (known enzymes and new enzymes provided by P5) and detailed enzyme characterization for the establishment of reaction kinetic models (P6). The Siebers's group has expertise in studying archaeal metabolism and characterization of diverse metabolic enzymes and pathways. The genetic tools for expression in Saci, construction of in frame markerless deletion mutants and ectopic integration of foreign DNA as well as methods for protein purification and the enzyme assays are established. P1 coordinated different Sulfolobus systems biology projects (SulfoSYS, SulfoSYS BIOTEC and HotSysAPP) and is experienced in data management and poly-omics data integration [1-3]. Evonik is one of the world's leading specialty chemicals companies. At Evonik, there is a special Life Cycle Management (LCM) team, which performs LCA to analyse the environmental impact of products and processes holistically. LCA (ISO 14040/14044) will be used to analyze the environmental impacts of the extremolytes starting from material and energy in- and outputs of all production steps via raw resource extraction up to the finished product, followed by the product use and finally the waste treatment or recycling. Up to 15 impact categories (e.g. climate change, water resource depletion, human toxicity, etc.) will be addressed and the most important impact sources (hot spots). production methods will be identified Federal Research Center of Biotechnology, Russian Academy of Sciences (FRCB), is a recently established association of three academic institutes, one of which is Winogradsky Institute of Microbiology, a world-known center of microbial diversity investigations. The team of Prof. Elizaveta A. Bonch-Osmolovskaya, the head of the Extremophiles Biology Department, during past 20 years is specializing in studies of extremophilic microorganisms, in majority thermophiles, by culture-dependent and independent (meta)genomic-based techniques .In the deposition of the FRCB team is the culture collection numbering several hundred novel extremophilic, mainly thermophilic, strains, enrichments and natural samples, as well as (meta)genomicdata, which will comprise a significant part of the platform for the screening of novel target genes within the project. FRCB as a Partner 5 (P5) will contribute to the work packages 2 and 3 (screening of novel target genes, genomic and metagenomics sequencing, genome analysis and data transfer). P5 is responsible for the screening of novel enzymes for extremolyte production in thermophilic habitats. The partner has massive (meta)genome data available that will be analysed by using advanced bioinformatics tools (eg. Toshchakov et al., 2015). Novel metagenomics libraries will be obtained to expand the dataset for the screening of enzymes for extremolyte synthesis. Novel thermophilic isolates available for P5 will be screened for the production of extermolytes using techniques and(or) materials, provided by other partners. The genomes of the isolates with positive target activity will be sequenced for further analysis of pathways for extremolytes biosynthesis and verification of candidates by expression in Escherichia coli, Thermus thermophilus and Sulfolobus acidocaldarius The ICRM in Milan Italy within the laboratory of Dr Daniela Monti will.in collaboration with other partners will be involved in the set-up of the recombinant expression of selected biocatalysts in E. coli. The genes coding for target enzymes will be cloned in suitable expression vectors and their production in soluble and active form will be compared under different expression conditions, e.g., in the presence of different medium and at different incubation temperatures. To overcomepossible limitations in enzyme recovery due to solubility issues, molecular chaperones-assisted expression will be tested, for example, byco-expression of GroEL/GroES complexes. Alternatively, to enhance protein expression and solubility, different fusion technologies, e.g., thatusing the small ubiquitin-like modifier (SUMO) protein, will be applied. The recombinant biocatalysts will be provided by suitable tags, e.g.,His-tag, to simplify the purification procedures and avoid the presence of contaminant enzymes for further studies. P3 will be involved also in the functional characterization of novel enzymes provided. In particular, biocatalysts performances will be evaluated under differen treaction conditions, with a special focus on those features that are expected to have a strong impact on their possible applicability in synthetic processes, such as thermostability, pH-activity profile, tolerance toward organic solvents, substrate and product inhibition. This partner has extensive experience in the optimization of coupled (chemo)enzymatic processes suitable to the efficient production of compounds of industrial and pharmaceutical interest (1-6). Therefore, in collaboration with P1 and P2, P3 will contribute to the development of either one-pot sequential or cascade multienzymatic systems aimed at the in vitro production of the new extremolytes. In both cases, the isolation of intermediates will be avoided, thus increasing the synthetic efficiency of the processes. When needed, for example in the case of the synthesis of di-myo-1,1'-inositol-phosphate (DIP), suitable cofactor regeneration systems will be employed to allow the use of catalytic amounts of the expensive nicotinamide coenzymes. In situ cosubstrate formation/coproduct removal systems will be applied as well toimprove the productivity of the bioconversions. The aim of the HotSolute project is to design two new thermophilic strains -Sulfolobus acidocaldarius (Saci) and Thermus thermophilus (Tth)- of archaeal and bacterial origin, respectively, for the production of three extremolytes: cyclic 2,3 di-phosphoglycerate (cDPG), di-myo-1,1'-inositol-phosphate (DIP) and mannosylglycerate (MG). These extremolytes are found in hyperthermophiles and cannot be produced in common mesophilic host so far. The pathways for solute formation have been well established and several of the enzymes have been characterized. For the construction and optimization of the designed strains we propose both an in vitro (enzyme cascades) and in vivo synthetic biology approach. The University of Stellenbosch will contribute a variety of enzyme kinetic data types to be will be generated: i.e. detailed enzyme kinetics for enzymes involved in the threesynthesis pathways after expression in Escherichia coli, Saci and Tth and enzyme activities in cell extracts of designed Saci and Tth strains. The role of the computational systems biology group in the proposal is to integrate the kinetic information in mathematical models that willbe validated with metabolomics data sets for pathway intermediates and final product analysis. Once the kinetic models are validated, theywill be analysed in silico to select the optimal conditions for maximal product formation rates. In addition, the computational systems biology group of Prof Snoep will assist in data and model management for the project using the SEEK platform (Wolstencroft et al., 2011) on the FAIRDOMHub (https://www.fairdomhub.org). This platform was developed for dedicated Systems Biology projects over the last decade and is now made publically available via the FAIRDOMHub. The platform makes it easy for research groups to share data, with access control, automated version control, ISA structure for linking data and model files in a structured way, and to link to external platforms or data sources. All models generated for the project will be made available as SBML files via the FAIRDOMHub and can be simulated in JWS Online, with dynamic linking to data files in the SEEK platform. The Systems Biology group of Prof Snoep has ample experience in building detailed kinetic models for glycolytic pathways in several different organisms, and he has actively advocated reproducible model construction and validation procedures. Prof Snoep has been involved with the SEEK development. |
| Impact | Multidisciplinary approach to use thermophilic bacteria and archaea as cell factories for the production of extremolytes |
| Start Year | 2018 |
| Description | Hotsolute Grant. Collaborations with Evonik,Germany, Prof Siebers, University of Essen, Germany, Dr vDaniela Monti, ICRM,Milan, Italy, Prof Elizaveta Bonch-Osmolovskaya,Russia |
| Organisation | Evonik Industries |
| Country | Germany |
| Sector | Private |
| PI Contribution | We have been involved in the expression of enzymes for extremolyte production from different sources in the T. thermophilus system and detailed enzyme characterization for the establishment of reaction kinetic models. We have worked closely with P1 and to integrate the optimized modules for cDPG, and DIP in Tth for strain design in collaboration with P1, P3 and P5. A paper on extremolyte production of cyclic 2,3 di-phosphoglycerate in T. thermophilus has been achieved published. Characterisation and Structural studies on the unique cDPGS enzyme has been carried out and a paper is currently being submitted to Frontiers in Microbiology. Enzyme cascade and strain optimization will be performed with the aid of modelling by Exeter in collaboration with P1 and P6. Exeter has expertise in using the bacterial Tth system as a thermophilic cell factory and has knowledge with both commercially available expression plasmids for this organism and construction of new plasmids including a Biobrick Tth /E.coli vector together with promotor design, codon optimization . This expertise has been achieved partly as part of a BBSRC Case studentship with GSK as part of and enzyme cascade developed for biocatalysis. Tth has an added advantage of being naturally competent due to a specialized macromolecular DNA translocator assembly which allows the entry of foreign DNA past the cell envelope into the cell. We have also collaborated with the partner in Italy and partner in Russia with bioinformatics screening to look for new enzymes for extremolyte synthesis found in other thermophilic genomes and metagenomes with P5 including the DNA resource from the EU Hotzyme project where P1,P3,P5 collaborating together. This has involved the cloning of the thermophilic genes related to the cDPG and 2PGK genes in E.coli and protein characterization with P3 and work with P1 on assay development. A variant of the 2PGK2 is currently being screened for crystallisation of this enzyme which produced only small crystals not suitable for X-ray characterisation from the original Methanococcus source. . The Exeter group has extensive experience in cloning thermophilic bacterial and archaeal enzymes and their industrial applications as summarized in two recent reviews and structural characterization . We have worked with P3 on development of the in vitro cascades of thermophilic enzymes involved in synthesis of other selected extremolytes and will investigate other pathways which could produce related products of industrial and pharmaceutical interest. Exeter and P3 have been part of a COST action on Systems Biocatalysis which has now finished. |
| Collaborator Contribution | The aim of HotSolute was to provide new extremolytes, i.e. cyclic 2,3 di-phosphoglycerate (cDPG), di-myo-1,1'-inositol-phosphate (DIP) and mannosylglycerate (MG), that cannot be produced in current mesophilic hosts by the use of thermophilic in vitro enzyme cascades and new thermophilic expression chassis. The thermophilic bacterium Thermus thermophilus (Tth) and the thermoacidophilic Archaeon Sulfolobus acidocaldarius (Saci) have been used as platform organisms. Studies using Sulfolobus as a thermophilic host have been carried out by the co-ordinator and further joint papers are in preparation. HotSolute has been coordinated by Bettina Siebers (P1, University of Duisburg-Essen, UDE) who was responsible for project and who has presented findings at on-line conferences with ERA-IB co-ordinators. Overall management of the project (e.g. organization of regular meetings, for all external reporting), for scientific and quality control of the project (e.g. internal reports) general adherence to data guidelines (SEEK, FAIRDOM), data standardization (SOPs)) and communication and dissemination (e.g. homepage, flyers) has been carried pout by partner 1 will help from all partners. The coordinator was assisted by Evonik industries (P4) in communication and dissemination aspects and by the Science Support Centre (SSC) at the UDE (e.g. consortium agreement, intellectual property rights). Scientifically, P1 has been involved in the expression of enzymes for extremolyte production from different sources in Saci (known enzymes and new enzymes provided by P5) and detailed enzyme characterization for the establishment of reaction kinetic models (P6). The Siebers's group has expertise in studying archaeal metabolism and characterization of diverse metabolic enzymes and pathways. The genetic tools for expression in Saci, construction of in frame markerless deletion mutants and ectopic integration of foreign DNA as well as methods for protein purification and the enzyme assays are established. P1 coordinated different Sulfolobus systems biology projects (SulfoSYS, SulfoSYS BIOTEC and HotSysAPP) and is experienced in data management and poly-omics data integration [1-3]. Evonik is one of the world's leading specialty chemicals companies. At Evonik, there is a special Life Cycle Management (LCM) team, which performs LCA to analyse the environmental impact of products and processes holistically. LCA (ISO 14040/14044) will be used to analyze the environmental impacts of the extremolytes starting from material and energy in- and outputs of all production steps via raw resource extraction up to the finished product, followed by the product use and finally the waste treatment or recycling. Up to 15 impact categories (e.g. climate change, water resource depletion, human toxicity, etc.) will be addressed and the most important impact sources (hot spots). production methods will be identified Federal Research Center of Biotechnology, Russian Academy of Sciences (FRCB), is a recently established association of three academic institutes, one of which is Winogradsky Institute of Microbiology, a world-known center of microbial diversity investigations. The team of Prof. Elizaveta A. Bonch-Osmolovskaya, the head of the Extremophiles Biology Department, during past 20 years is specializing in studies of extremophilic microorganisms, in majority thermophiles, by culture-dependent and independent (meta)genomic-based techniques .In the deposition of the FRCB team is the culture collection numbering several hundred novel extremophilic, mainly thermophilic, strains, enrichments and natural samples, as well as (meta)genomicdata, which will comprise a significant part of the platform for the screening of novel target genes within the project. FRCB as a Partner 5 (P5) will contribute to the work packages 2 and 3 (screening of novel target genes, genomic and metagenomics sequencing, genome analysis and data transfer). P5 is responsible for the screening of novel enzymes for extremolyte production in thermophilic habitats. The partner has massive (meta)genome data available that will be analysed by using advanced bioinformatics tools (eg. Toshchakov et al., 2015). Novel metagenomics libraries will be obtained to expand the dataset for the screening of enzymes for extremolyte synthesis. Novel thermophilic isolates available for P5 will be screened for the production of extermolytes using techniques and(or) materials, provided by other partners. The genomes of the isolates with positive target activity will be sequenced for further analysis of pathways for extremolytes biosynthesis and verification of candidates by expression in Escherichia coli, Thermus thermophilus and Sulfolobus acidocaldarius The ICRM in Milan Italy within the laboratory of Dr Daniela Monti will.in collaboration with other partners will be involved in the set-up of the recombinant expression of selected biocatalysts in E. coli. The genes coding for target enzymes will be cloned in suitable expression vectors and their production in soluble and active form will be compared under different expression conditions, e.g., in the presence of different medium and at different incubation temperatures. To overcomepossible limitations in enzyme recovery due to solubility issues, molecular chaperones-assisted expression will be tested, for example, byco-expression of GroEL/GroES complexes. Alternatively, to enhance protein expression and solubility, different fusion technologies, e.g., thatusing the small ubiquitin-like modifier (SUMO) protein, will be applied. The recombinant biocatalysts will be provided by suitable tags, e.g.,His-tag, to simplify the purification procedures and avoid the presence of contaminant enzymes for further studies. P3 will be involved also in the functional characterization of novel enzymes provided. In particular, biocatalysts performances will be evaluated under differen treaction conditions, with a special focus on those features that are expected to have a strong impact on their possible applicability in synthetic processes, such as thermostability, pH-activity profile, tolerance toward organic solvents, substrate and product inhibition. This partner has extensive experience in the optimization of coupled (chemo)enzymatic processes suitable to the efficient production of compounds of industrial and pharmaceutical interest (1-6). Therefore, in collaboration with P1 and P2, P3 will contribute to the development of either one-pot sequential or cascade multienzymatic systems aimed at the in vitro production of the new extremolytes. In both cases, the isolation of intermediates will be avoided, thus increasing the synthetic efficiency of the processes. When needed, for example in the case of the synthesis of di-myo-1,1'-inositol-phosphate (DIP), suitable cofactor regeneration systems will be employed to allow the use of catalytic amounts of the expensive nicotinamide coenzymes. In situ cosubstrate formation/coproduct removal systems will be applied as well toimprove the productivity of the bioconversions. The aim of the HotSolute project is to design two new thermophilic strains -Sulfolobus acidocaldarius (Saci) and Thermus thermophilus (Tth)- of archaeal and bacterial origin, respectively, for the production of three extremolytes: cyclic 2,3 di-phosphoglycerate (cDPG), di-myo-1,1'-inositol-phosphate (DIP) and mannosylglycerate (MG). These extremolytes are found in hyperthermophiles and cannot be produced in common mesophilic host so far. The pathways for solute formation have been well established and several of the enzymes have been characterized. For the construction and optimization of the designed strains we propose both an in vitro (enzyme cascades) and in vivo synthetic biology approach. The University of Stellenbosch will contribute a variety of enzyme kinetic data types to be will be generated: i.e. detailed enzyme kinetics for enzymes involved in the threesynthesis pathways after expression in Escherichia coli, Saci and Tth and enzyme activities in cell extracts of designed Saci and Tth strains. The role of the computational systems biology group in the proposal is to integrate the kinetic information in mathematical models that willbe validated with metabolomics data sets for pathway intermediates and final product analysis. Once the kinetic models are validated, theywill be analysed in silico to select the optimal conditions for maximal product formation rates. In addition, the computational systems biology group of Prof Snoep will assist in data and model management for the project using the SEEK platform (Wolstencroft et al., 2011) on the FAIRDOMHub (https://www.fairdomhub.org). This platform was developed for dedicated Systems Biology projects over the last decade and is now made publically available via the FAIRDOMHub. The platform makes it easy for research groups to share data, with access control, automated version control, ISA structure for linking data and model files in a structured way, and to link to external platforms or data sources. All models generated for the project will be made available as SBML files via the FAIRDOMHub and can be simulated in JWS Online, with dynamic linking to data files in the SEEK platform. The Systems Biology group of Prof Snoep has ample experience in building detailed kinetic models for glycolytic pathways in several different organisms, and he has actively advocated reproducible model construction and validation procedures. Prof Snoep has been involved with the SEEK development. |
| Impact | Multidisciplinary approach to use thermophilic bacteria and archaea as cell factories for the production of extremolytes |
| Start Year | 2018 |
| Description | Attendance and invited oral presentation at International Extremophiles Meeting, Greece September 2022 by Dr Simone De Rose postdoc |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | International Scientific Meeting |
| Year(s) Of Engagement Activity | 2022 |
| Description | Invited Plenary Lecture and Best Poster Prize by postdoc Dr Simone De Rose International Thermophile, Japan |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Industry/Business |
| Results and Impact | Plenary Lecture at International Meeting |
| Year(s) Of Engagement Activity | 2019 |
| Description | Kick off Event of ERA-CoBiotech june 2018 Frankfurt, Germany |
| 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 | Introduction of funded ERA-CoBiotech successful projects funded to start in 2018 to a general audience at large industrial fair Achema in Frankfurt, Germany |
| Year(s) Of Engagement Activity | 2018 |
| Description | MECP 2020+ Meeting September 2021 Virtual due to COVID |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Poster presentation Dr Simone De Rose |
| Year(s) Of Engagement Activity | 2021 |
| URL | https://mecp2020.com |
| Description | Poster Presentation at Novel Enzymes Conference, Germany March 2023 |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Poster presentation Novel Enzymes Involved in the Synthetic Pathway for the Extremolyte Cyclic Diphosphoglycerate. Simone A. De Rose, Nicholas J. Harmer, Michail Isupov and Jennifer A. Littlechild |
| Year(s) Of Engagement Activity | 2023 |