DNA Helix

Cofund on Biotechnologies

Innovation for Europe – life science meets market application

First transnational call for research projects

Aim of the co-funded call

The first transnational joint call for research projects (co-funded by the EC) within the framework of ERA CoBioTech aimed at biotechnology as a key enabling technology (KET), in the context of the bio-based economy to tackle 21st century societal challenges such as decarbonisation of the economy and reduction of the reliance on fossil feedstocks. Funding is granted for a maximum of three years according to national regulations.

Procedure

In a two-phase submission process 100 eligible pre-proposals were peer reviewed and ranked by a panel of international experts from 15 different countries. Based on the ranking and the available national/regional funding 41 research consortia were invited to submit a Full Proposal. After peer review and ranking of the Full Proposals by the same evaluation panel the Call Steering Committee selected 22 research consortia for funding from the ranking list again, within the limits of available national/regional and EC funding.

 

Timeline

  • pre-proposal: the submission deadline was by March 2, 2017
  • full proposal: the submission deadline for invited proposals was by July 20, 2017.

Call documents can be found here

Selected projects recommended for funding:

BestBiosurf

PROJECT PARTNERS:

Contact Name of Organization Country
Philippe JACQUES (coordinator)
Contact: philippe.jacques[at]uliege.be
University of Liège. Belgium
Marnix MEDEMA Wageningen University & Research Netherlands
Hugo GRAMAJO Instituto de Biología Molecular de Rosario IBR-CONICET-UNR Argentina
Harald GROSS Eberhard Karls University of Tuebingen (EKUT), Pharmaceutical Institute Germany
Arnaud DELECROIX LIPOFABRIK Belgium Belgium
John Belk Dasic International Ltd United Kingdom

Project summary

Biosurfactants can be defined as surface-active biomolecules produced by microorganisms with wide-range of applications such as emulsification, detergency, solubilization, lubrication and phase dispersion. In recent years, these biomolecules have attracted wide interest from the scientific community but also from many industrials due to their unique properties like specificity, low toxicity and relative ease of preparation. Also, biosurfactants show several advantages over chemical surfactants, including higher biodegradability and better environmental compatibility.

In light of all of this, the BestBioSurf project aims at producing novel and eco-friendly biosurfactants in a cost-effective manner through initial pilot validation in laboratory settings (TRL 4), to a larger bio-process scale up (TRL 5). In order to do so, the BestBioSurf project will use the Bacillus subtilis bacterial host system as a primary choice for producing these novel biosurfactants. This is mainly due to the strain’s long history and well-established experimental proof-of concept use in the biotechnological area of lipopeptide bioengineering (TRL 3).

Precisely, we introduce a novel and original strategy based on bioinformatics, synthetic biology and metabolic engineering to perform molecular diversification of well establishedbiosurfactants with high efficacy, low toxicity and high biodegradability, and to produce these at high titers. First, toxicity/efficacy assays with existing lipopeptide and molecular modeling of chemical variants of these compounds with target liquids will be used to define the physicochemical properties of the best biosurfactant to produce. Then, genome mining and synthetic biology will enable the identification and the construction of novel biosynthetic pathway variants towards the target compounds. To reduce their cost price, fatty acid and amino acid metabolism in the producing cells will subsequently be genetically optimized in order to turn very cheap co-substrates provided by a bioethanol industrial producer into high product titers. Finally, the newly produced (novel) surface-active compounds will be checked for their efficacy and ecotoxicity by the end-user study of this project. This end-user is an SME close to the market that could replicate widely the technology through its various activity areas including oil dispersants, innovative chemical cleaning, hygiene, aerospace to rail rolling stock and waterborne pollution. Many other potential applications with high eco-friendly impacts include agriculture (biosurfactants-based biopesticides), medicine (anti-microbial & anti-cancer activity), and cosmetics (emulsification & foaming properties). This wide range of applications opens up a whole new window of opportunities for partnering in the future with many more end-users that could benefit from the BestBioSurf technologies.

Bioprocesses for the optimized, integrated production of butyl esters from sustainable resources

PROJECT PARTNERS:

Contact Name of Organization Country
Alexander Wentzel (coordinator)
Contact: alexander.wentzel[at]sintef.no
SINTEF Materials and Chemistry (SINTEF) Norway
Peter Dürre Ulm University (UULM) Germany
Liz Jenkinson Green Biologics Ltd. (GBL) United Kingdom
Olaf Wolkenhauer University of Rostock (UROS) Germany
Hector OSUNA PROCESSIUM (PROC) France
Jeremy Woods Imperial College London (ICL) United Kingdom

Project summary

Industrial Biotechnology is a key enabling technology to produce a plethora of different bio-based products from sustainable resources and a driver for developing the bio-based economy in Europe. Systems biology and Synthetic biology are recent additions to the biotechnology toolbox that in interplay with bioprocess and chemical process technology can help develop competitive industrial bioprocesses for manufacturing valuable novel products.

The specialty chemical industry is a $450 billion market, and is an increasingly important part of the $5.4 trillion global chemical market. Within this market, butyl esters, derivable from n-butanol (BuOH) and suitable organic acids by esterification, have diverse uses as commodity chemicals and drop-in biofuels, but also represent high value opportunities within the fragrance and flavour industry, cosmetics, specialty polymers and coatings.

The production of BuOH in the anaerobic clostridial ABE fermentation process is well established, achieving commercial scale in the US in 2016 by UK-based company and BESTER project partner Green Biologics Ltd. (GBL). What is needed to produce butyl esters for the commodity market are efficient processes to produce suitable organic acids from renewable resources as counterparts for BuOH in catalytic esterification. In combination, the use of esterase enzymes as natural, sustainably producible biocatalysts for ester formation will allow entirely green bioprocesses for the production of different butyl esters, thus increasing market value of the ABE process and reducing GHG emissions.

The ERA-CoBioTech project BESTER establishes clostridial bioprocesses for an optimized integrated production of a range of different butyl esters using lignocellulosic sugars as a sustainable feedstock for the commodity chemicals market. Efficient organic acid production processes will be developed, which can be linked to ABE fermentation processes as a source of BuOH. BESTER will thereby address the two key handles for efficient production of the three acids, i.e. a) Systems biology driven strain engineering using Synthetic biology principles to establish new acid production in suitable clostridial chassis strains and mitigate key metabolic bottlenecks hampering high productivity, and b) smart process design and integration to prevent inhibitory effects of the acids produced and the BuOH added in efficient bioprocesses with continuous enzymatic ester product formation and recovery. Lignocellulosic feedstock hydrolysate will be used as the main feedstock in BESTER. The primary project deliverable will be a set of scalable, robust, and highly productive manufacturing processes for selected butyl esters.

The BESTER project connects six project partners with highly complementary expertise from four different ERA-CoBioTech partner countries, i.e. Norway, UK (2), Germany (2), and France, in order to maximise project output and share risks, costs and skills. The project is substantially industry-driven with large enterprise GBL (UK) and SME Processium SA (FR) participating as active partners. Furthermore, Borregaard AS (NO) will support the project by providing both expert advice from the project's Advisory Board and fermentation feedstock. The three companies are positioned along the butyl ester value chain, which will ensure that project results are efficiently taken further towards commercialization. BESTER will help to tackle the 21st century societal challenge of reducing our reliance on fossil feedstocks for chemical production and to achieve sustainable industrial development in Europe.

Methyl Transferases for the Functional Diversification of Bioactives

PROJECT PARTNERS:

Contact Name of Organization Country
Ulf Hanefeld (coordinator)
Contact: u.hanefeld[at]tudelft.nl
Delft University of Technology Netherlands
Michael Richter Fraunhofer IGB Germany
Helen Hailes University College London United Kingdom
Jennifer Andexer University of Freiburg Germany
Shimon Bershtein Ben-Gurion University of the Negev Israel
Murray Brown GSK United Kingdom
Elizabeth Lewkowicz Universidad Nacional de Quilmes Argentina

 Project Summary

BioDiMet aims to implement Nature’s strategy to selectively methylate target compounds as a robust enzymatic platform ready for use at an early industrial scale. In Nature enzymatic methylation by SAM-dependent methyltransferases is a key step to accomplish and enhance bioactivity. The reactions can occur in a regioselective and/or stereoselective manner and comprise outstanding examples that cannot be realised by common synthetic methods under similar conditions.

This is of great relevance to the need for the development of novel bioactives in multi-billion € markets of the pharma, agrochemical and fragrance/flavor industries. In this area key to the structural diversity and bioactivity of many compounds containing amine and alcohol functionalities is selective methylation. This is very difficult to achieve using established chemical synthetic methods where mainly toxic methylation agents such as methyl iodide and dimethyl sulfate are applied. Enzymatic approaches have the advantages of sustainability, the avoidance of toxic reagents, and regiospecificity. For some substrates enantioselectivities may also be achieved.

Despite the importance in Nature, SAM-dependent methyltransferases are completely underexploited for use in industrial synthesis. In the past, the reasons for this were mainly because SAM cofactor supply/recycling as well as a robust methyltransferase toolbox were not available. In recent years members of the BioDiMet consortium demonstrated that these limitations do not hold true anymore by establishing smart enzymatic methylation cascades with integrated SAM supply or recycling.

BioDiMet is now the subsequent and necessary next step towards industrialisation of the enzymes in the pharma industry. The activities will be carried out under the roof of 3 technology platforms (TPs) and cover the synthesis of target molecules for methyl derivatisation, the development of novel enzymes for methylation and also for providing prospective SAM alkylation analogues. It also involves the development of methylation cascades with cofactor supply and the optimization of enzymatic steps. Given the essentially irreversible character of the methylation, the MT catalysed reactions will be utilized to shift the thermodynamic equilibria of preceding reactions in the reaction cascades.

Development of scaled-up reactions and down-stream processing methods will facilitate the industrial feasibility of the platform. In conclusion BioDiMet aims to extend and improve known and innovative methyltransferase reaction systems to a significantly advanced level. This involves the identification of novel substrates, enzymes and tailored enzyme cascades including the synthesis of unique target compounds that potentially can be methylated at an early industrial scale.

BioDiMet unites leading partners from academia, research institutions and pharma industry in the areas of i) synthetic biology for methyl transfer cascade design, ii) enzymatic and chemoenzymatic synthesis (biotechnology and chemistry) for the purpose of producing added value chemicals starting mainly from natural resources or biobased molecules. Equally iii) bioinformatics, and also iv) available data from systems biology will be considered for the discovery and optimisation of enzymes in order to shift the emerging and highly promising enzymatic methylation technology from TRL3-4 to TRL5-6.

Sustainable Production of Added Value Chemicals from SynGas-derived Methanol Through Systems
and Synthetic Biology Approaches

PROJECT PARTNERS:

Contact Name of Organization Country
Nigel Minton (coordinator)
Contact: nigel.minton[at]nottingham.ac.uk
University of Nottingham United Kingdom
Peter Dürre Ulm University Germany
Volker Müller Goethe University Frankfurt Germany
Philippe Soucaille INSA, University of Toulouse, LISBP France

Project summary

One of the greatest challenges facing society is the future sustainable production of chemicals and fuels from non-petrochemical resources while at the same time reducing greenhouse gas emissions. The recalcitrance of lignocellulose to deconstruction for feedstock purposes is making the economic development of biologically-based processes extremely challenging. This has led to the concept of using low-cost, abundant one-carbon (C1) feedstocks. Here the focus has been on using C1 gases, such as CO/CO2 and CH4, sourced as a waste from industrial processes, anaerobic digestion or deliberately formed as synthesis gas (syngas) through the gasification of any waste containing biomass (agricultural/ forestry residues and municipal solid waste) or by the reformation of shale gas. Such an approach is not without its issues. The mass transfer of gases into the liquid phase in reactors places constraints on reactor design and performance, while in the case of aerobic chassis the additional presence of H2, and O2 is potentially explosive. In contrast, as a liquid, methanol does not suffer from mass
transfer issues in fermenters and is more easily stored and transported. It can be made from many sustainable feedstocks, including biomass, MSW, biogas, waste CO2, and even renewable electricity.

The case for using methanol as a feedstock to make chemicals and fuels is, therefore, compelling. In this project, BIOMETCHEM, we will exploit the progress made in developing effective genetic systems for Eubacterium limosum to derive engineered strains able to produce the value-added products γ-aminobutyric acid, GABA, (useful in the pharmaceutical and food additive industries) and 1,4-butanediol, BDO (a platform chemical), from biomass derived methanol. Process strain will be derived through a combination of interdisciplinary methodologies, including systems biology (INSA, University of Toulouse), synthetic biology (UNOTT, University of Nottingham), metabolic engineering (ULM, University of Ulm), enzymology (UFRA, University of Frankfurt), and methanol fermentation development (All Partners). Responsible Research Innovation (RRI) practices will be embedded within the programme of work through the participation of dedicated Social Scientists from the Synthetic Biology Research Centre (SBRC) at Nottingham. Life Cycle Analysis (LCA) and Techno-Economic Analysis (TEA) will be undertaken by Nottingham in partnership with Johnson Matthey. BIOMETCHEM will lead to the development of new Sustainable production
and conversion processes based on methanol feedstocks derived from gasified (syngas) biomass residues and wastes or from industrial by-products (FT waste streams). This will lead to new value-added products, GABA and BDO, useful in the pharmaceutical/food additive industries and the chemical industry, respectively.

Ultimately, the developments made will lead to new sustainable industrial processes. Moreover, by combining resources and expertise, BIOMETCHEM connects research partners in three different countries (France, Germany and UK) with different but complementary scientific and technological expertise, thereby maximising resources and sharing the risks, costs and skills. The participants represent some of Europe’s leading experts in anaerobic metabolism, and in particular C1 feedstocks. The amalgamation of their expertise and resources provides the critical mass needed to compete with the rest of the world, in particular the US and China, where there is considerable activity in the C1 field.

Microbial conversion of C1 to value-added products by integrated systems and synthetic biology

PROJECT PARTNERS:

Contact Name of Organization Country
Trygve Brautaset (coordinator)
Contact: trygve.brautaset[at]ntnu.no
Norwegian University of Science and Technology Norway
Volker F Wendisch Bielefeld University Germany
Stephanie Heux INSA - Institut National des Sciences appliquées de Toulouse France
Oskar Zelder BASF SE Germany
Ingemar Nærdal SINTEF Materials and Chemistry Norway
Gregor Kosec Acies Bio d.o.o. Slovenia

Project summary

Society urgently needs a sustainable production processes for key platform chemicals with various applications in several industries, in light of resource scarcity. Developing a bioeconomy is a cornerstone to meet this grand challenge. The bioeconomy can make major socioeconomic contributions, lead to health improvement, boost the productivity of agriculture and industrial processes, and enhance environmental sustainability.

The C1Pro project aims to establish a sustainable platform for methanol-based production of value-added products (GABA,
5AVA, L-Pro and L-PA) with proven industrial applications. Methanol is an attractive and alternative raw material for biotechnological processes because of its chemical properties, relatively low price and availability from both fossil and renewable sources. The Gram-positive, methylotrophic and thermophilic bacterium Bacillus methanolicus was chosen as model organism in this project for several reasons: it utilizes methanol as raw material for growth and energy, it grows at elevated temperatures (50 – 55 °C), it naturally overproduces L-glutamate, and its classical mutants have demonstrated a high potential to overproduce L-lysine. We propose to apply innovative technologies in the fields of modelling, development of genetic tools, strain engineering, fermentation technology and downstream processing to establish and develop methanol-based production.

Four different products, GABA, 5AVA, L-Pro and L-PA, were chosen deliberately as they share a) biosynthesis pathways, b) functional characteristics and c) industrial applications. The omega-amino acids GABA and 5AVA can be cyclized by lactamization, while L-Pro and L-PA are cyclic amino acids. Biosynthesis of L-Pro and GABA starts from L-glutamate, whereas L-PA and 5AVA derive from L-lysine. The targeted products serve as building blocks of polymers or precursors of pharmaceuticals and other biologically active substances. The commonalities of the chosen product pairs GABA/5AVA and L-Pro/L-PA enable transferability: process intensification and DSP development for one example (GABA) can be carried out in parallel with initial strain and fermentation development for the other products. Later in the project, the most promising processes will be scaled up.

Systems and synthetic biology approaches are key to the proposed strain and process development, which is facilitated by common biosynthesis pathways. Novel genetic tools will simplify regulated gene expression on different levels via CRISPR/Cas9 for genome modifications and riboswitches for regulatory circuits. Pathway design will be guided by the genome-scale metabolic model which will be iteratively fine-tuned based on experimental test results. Strain performance in methanol-based fermentations will be characterized in-depth by RNAseq, metabolome and/or flux analyses. This will guide iterative optimization of strains and fermentation conditions in subsequent rounds.

All data will be collected in an LCA-compliant way and used to select the most promising strain(s) for up-scaling where methanol-based production at 20-150 L scale will be performed in a clean bioprocess. Finally, GABA isolation to at least 80% purity will be demonstrated.

Computation for Rational Design: From Lab to Production with Success

PROJECT PARTNERS:

Contact Name of Organization Country
Henk Noorman (coordinator)
Contact: H.J.Noorman-9[at]tudelft.nl
Technical University Delft Netherlands
Ralf Takors University of Stuttgart Germany
Frank Delvigne University of Liège Belgium
Philippe Gabant SYNGULON Belgium
Emile van de Sandt DSM Sinochem Pharmaceuticals Netherlands

Project summary

The bioprocess industry needs new efficient and sustainable routes to manufacture bio-products. Bioprocesses use the power and versatility of nature via microorganisms that make bio-products from renewable feedstocks. Micro-organisms can very well be engineered as efficient cell factories. However, the gap between the cell environment at lab and production scales is causing gross resource and asset utilization inefficiencies, and is a barrier to fast and successful scale-up. This bottleneck has become one of the most prominent risks for the bio-economy, moving from innovation (supported by the revolutions in metabolic engineering and molecular biology) to commercialization.

Today, scale-up in industry is still driven by physical guidelines or heuristic approaches but does not consider individual cellular properties for ab initio and in silico design. Previous pioneering studies could not exploit the full predictive potential because details of metabolic and transcriptional regulatory responses were not yet known and computational capacities limiting.

In recent years, advanced tools have been developed which, taken together, offer a way out from this innovation ‘valley of death’. This combines systems biology, synthetic biology, bioinformatics and bioprocess development.

The project will develop a robust and model-based simulation platform to ramp up the scale-up techniques driven by profound biological and physical understanding. This individual and integrated tool will enable higher level of understanding about the fermentation process, both in the fluid dynamics (physics) and metabolic dynamics (biology), and in their mutual connection.

In essence, the project aims to replace conventional, empirical scale-up criteria by predictive modeling and rational design, based on combined biological and physical expertise. The work will focus on two leading industrial cases, comprising the micro-organisms Penicillium chrysogenum and Saccharomyces cerevisiae, but has general implications.

The different parts of the platform will be developed by the three academic partners. Then, the platform will be used to assess the Syngulon technology for controlling the phenotypic state of the microbial population, relevant to large-scale performance, based on synthetic biology methods. Finally, DSM as principal end-user of the technology will benefit from the platform to enhance their fermentation process development capability.

Development and application of computational approaches to design better scale-down simulators, enable faster scale-up, and improve the energy and resource efficiency of fermentations, will accelerate bringing bio-innovations to the markets. This bio-based drive is further essential to help solving the mega-issues of climate change, food security and energy supply. The integrated computational solution proposed will contribute to reconciliation of two main, competing, business success factors: speed and quality.

The project aims at:

  • accelerating the bioprocess development including plant start-up by at least 20% (e.g. from 5 to 4 years), which could after 10 years further develop into a factor 5 (from 5 to 1 year).
  • developing guidelines for constructing strains such that they are robust enough for harsh production conditions
  • the early identification of scale-up sensitive properties of novel producers to ensure efficient use of manpower and research capacities
  • reducing of the order of 10’s of tonnes of CO2 emission per large-scale fermentation
  • reducing the energy requirements of at least 10%, i.e. in the order of 10 MWh, per run executed on industrial scale
  • reducing by at least 20% the development budget (e.g. from 10 M€ to 8 M€), which could after 10 years further advance to a factor 5 (from 10 to 2 M€)

* Assuming analogy of key findings of E. coli with S. cerevisiae and P. chrysogenum.

Model-guided evolution for balanced attenuation of wine ethanol content by developing non-GMO yeast strains and communities

PROJECT PARTNERS:

Contact Name of Organization Country
Ramon Gonzalez (coordinator)
Contact: rgonzalez[at]icvv.es
Consejo Superior de Investigaciones Científicas (CSIC) Spain
Albert Mas Universitat Rovira i Virgili Spain
Eivind Almaas NTNU - Norwegian University of Science and Technology Norway
Esperanza Tomás Bodegas Roda S.A. Spain
Jonas Warringer University of Gothenburg other (Sweden)
Kiran Patil European Molecular Biology Laboratory Germany

Project summary

Biotechnological problem

Increasing temperature in the European wine producing regions is having a negative impact on this key sector. Climate change results in a lack of balance between technological and phenolic ripening of wine grapes and, as a consequence, alcohol increase in wines. This trend is of great concern for the European wine industry because it has a negative impact on wine quality, becomes a hurdle for international trade, and jeopardizes compatibility of moderate wine consumption with a healthy lifestyle.

CoolWine Strategy

We propose a two-track strategy to reduce ethanol yield during wine fermentation (Figure1). Track 1: model-guided adaptive laboratory evolution of wine yeasts. Track 2: model-guided assembly of improved communities including S. cerevisiae as well as alternative yeast species.

Scientific approach

CSIC partner has previously successfully used microbial consortia and oxygenated fermentations to reduce ethanol content of wines. Currently, several companies and research groups are also trying to follow this path, thus endorsing the technological and commercial validity of the approach. Although the current results are encouraging, we have also identified some bottlenecks (e.g. increased acetate production that is harmful for wine quality, see TRL description for details). In order to overcome these hindrances, we will tackle both applied and basic scientific challenges.

For developing improved wine yeasts through model-guided ALE, currently available metabolic models and computational tools will be improved in order to account for data concerning respiro-fementative balance and acetic acid production. Computational models will be further informed by experimental data from different yeast mutants. In terms of the community models, we will identify metabolic pathways to be positively or negatively selected for in each species during ALE. This will allow us to develop microbial consortia suitable for alcohol level reduction. The EvolveX algorithm (partner EMBL, patent filed in July 2016) will be used to design experimental conditions for ALE. For the design of yeast consortia and identification of target pathways to improve them we will integrate “omics” data to SMETANA. We will then run massive-scale ALE in order to explore a number of experimental conditions, and to cope with the stochastic nature of spontaneous genetic mutation.

Excellence

CoolWine will develop an innovative solution by using, improving, and extending to other species, the technology platform established under WineSys (an ongoing ERA-SysApp project). The CoolWine team spans scientific and industrial expertise in wine yeast physiology, metabolic modelling, “omics” analysis, massively parallel adaptive evolution, oenology, and social sciences. The team members have a successful track record of working together and are involved in several collaborative projects.

Impact

The companies involved in this project (Roda, Torres, and Agrovin) have clearly identified their consumers’ demand for solutions to high ERA CoBioTech alcohol content. Achievement of higher TRLs will be straightforward from the expected results of CoolWine. This is because the technological steps and scientific approaches used in the project are devised to be very close to the actual production. The strains and communities developed will be tested at commercially relevant level. Indeed, the contribution of Roda in WP5 will be decisive to reach relevant results. They have a strong position in their cognate sectors, and a long culture of research and innovation.

Production of wines with reduced ethanol content using GMO-free solutions will strengthen the market position of European wines. We also expect CoolWine outcomes to contribute to healthier moderate wine consumption.

To ensure the highest social impact for this project, we have included WP6. Results from this WP will help CoolWine partners modulate biotechnological targets, and develop a well-designed communication strategy.

Fabrication of hierarchically organized multi-functional heterogeneous biocatalysts for the modular synthesis of ω-amino acids from renewable feedstocks

PROJECT PARTNERS:

Contact Name of Organization Country
Aitziber L. Cortajarena (coordinator)
Contact: alcortajarena[at]cicbiomagune.es
CIC biomaGUNE Spain
Ricardo Madrid Bioassays Spain
Francesca Paradisi University of Notthingham United Kingdom
Frank Schulz Ruhr-Universität Bochum Germany

 Project summary

Chemical synthesis catalyzed by enzymes is contributing to establish a modern chemistry supported on cleaner, faster and safer chemical reactions. In particular, cell-free metabolic engineering (or systems biocatalysis) in solution is currently emerging as an attractive alternative to synthetic biology using whole cells because isolated enzymes do not present regulation constraints at genomic level and the intensification of the chemical fluxes do negligible effect on the system subsistence. However, it presents major issues in terms of both process- and cost-efficiency because these soluble systems often reach low chemical yields, are notably unstable and their re-usability is rather limited. In order to overcome these limitations, this proposal aims to assemble multi-enzyme systems at the nanoscale of solid and porous materials aided by protein scaffolds that guarantee the hierarchical and spatial organization of the functional modules. This immobilized multi-enzyme cascade will be utilized as heterogeneous multi-functional biocatalyst to transform renewable raw materials into ω-amino acids in one-pot and with in situ cofactor regeneration. The fabrication and exploitation of such heterogeneous biocatalysts will be achieved by i) engineering the proposed artificial pathway for the synthesis of ω-amino acids using bio-oils and fermented diols as raw materials, ii) engineering a modular protein as scaffolding unit to organize the multi-enzyme system at the nanoscale, iii) in vitro assembling of multi-enzyme systems onto the protein scaffolds, iv) immobilizing such multi-enzyme assemblies on solid particles, v) using the hierarchically organized multi-functional heterogeneous biocatalysts for the efficient and sustainable production of long and short ω-amino acids vi) 10 L scaling-up the process under industrially relevant conditions and vii) manufacturing of one modular kit to produce multi-enzyme systems employing common laboratory methods and one modular kit that allows the solid-phase assembly of different functional modules based on scaffolded multi-enzyme systems. These new platforms will open an innovative tool to build sustainable pathways for chemical manufacturing of high added value molecules using renewable raw materials. The rational integration of different enzymes as functional modules with an engineered protein scaffold and a porous material as heterogeneous chassis will be addressed by combining protein engineering, surface chemistry and protein immobilization tools. The research team presents a solid and multidisciplinary background in those areas, which fully qualifies this team to carry out this project at the frontier between the chemistry and the biology. Moreover, the research consortium is hosted in different research institutions that provide a suitable research environment (three  academic partners; CIC biomaGUNE, University of Nottingham and Ruhr Universität-Bochum, and one industrial partner: Bioassays) to successfully address the major challenges and meet the main objectives of this project.

Thermophilic bacterial and archaeal chassis for extremolyte production

PROJECT PARTNERS:

Contact Name of Organization Country
Bettina Siebers (coordinator)
Contact: bettina.siebers[at]uni-due.de
University of Duisburg-Essen Germany
Jennifer Littlechild University of Exeter United Kingdom
Daniela Monti Consiglio Nazionale delle Ricerche Italy
Felix Müller Evonik Industries AG Germany
Elizaveta Bonch-Osmolovskaya Federal Research Center of Biotechnology, Russian Academy of Sciences Russia
Jacky Snoep Stellenbosch University other (South Africa)

Project summary

Background- 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 temperature, organic solvents and proteolysis. 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, 65-75°C, pH 7.0) and the thermoacidophilic archaeon Sulfolobus acidocaldarius (Saci, 75-80°C, pH 2-4), as new thermophilic, bacterial and archaeal platforms for the production of novel high added-value products, i.e. ‘extremolytes’. Extremolytes are small molecular compatible solutes found naturally in the cells of thermophilic species that accumulate in the cell in response to multiple environmental stresses and stabilize cellular components (including proteins, 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 (i.e. yeast, E. coli) is currently hampered by the hyperthermophilic origin of the respective metabolic pathways requiring 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 (meta)genome searches and 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 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. The project aims will be achieved by a multidisciplinary team of scientists and experts, who are leaders in their respective fields being brought together and working with associated SMEs and one major international company. Notably, the (trans)national core group is well established in extremophile research, biocatalysis and both Thermus and Sulfolobus biology providing an excellent starting point for this ambitious proposal.

This project will develop the current applications of thermophilic enzyme cascades and micro-organisms for the industrial production of small molecule extremolytes which have both medical and healthcare applications.

Biotechnological production of sustainable indole

PROJECT PARTNERS:

Contact Name of Organization Country
Dirk Bosch (coordinator)
Contact: dirk.bosch[at]wur.nl
Wageningen Plant Research Netherlands
Kristina Gruden National Institute of Biology Slovenia
Volker Wendisch Bielefeld University Germany
Vitor Martins dos Santos Wageningen University Netherlands
Peter van der Schaft Axxence GmbH Germany

Project summary

Our world is changing fast! Key global trends are rapid urbanisation, growing and ageing populations, and increased prosperity. This results in depletion of natural and petrochemical resources and climate change, which affects the quality of the environment and people's lives. Therefore, developing a bio-based economy is key to sustain our planet in the long term. Raw materials will have to be recruited from renewable sources.

Industrial biotechnology is potentially a very powerful technology in the transition from petrochemical to renewable resources. It uses microorganisms (fermentation) and enzymes (biocatalysts) to convert low-value agricultural residues which are not suitable to serve as foodstuffs into high value products. Microorganisms have the capacity to produce large amounts of building blocks from biomass by their endogenous metabolism. In addition, metabolic engineering is used to construct microorganisms containing cascades of enzymes that make high-value, high-purity, renewable chemicals.

Indole is a good example where biotechnological production can make a difference. Indole is an important flavouring compound with the main market in dairy, tea drinks and fine fragrances. It exhibits a floral odor typical for jasmine teas. There is a strong demand for a more cost-efficient and sustainable method for preparing natural indole. Synthetic indole, derived from coal tar, is available at moderate prices, while the highest quality food-grade indole (natural indole) is very highly priced, due to an expensive chemical conversion process. A reduction in production costs will most likely lead to an increased demand from the market.

The INDIE project aims to produce indole via microbial fermentation and enzymatic bioconversion. The production system will be inspired on indole produced in nature. Indole is produced in traces by a variety of bacteria as a signaling molecule. It can also be found at low concentrations in plant essential oils (e.g. jasmine oil). In bacteria and plants, indole is derived from the L-Trp biosynthetic pathway. In our project, we will use the bacterium C. glutamicum for the production of indole because it is capable of producing high amounts of L-Trp.

The approach to achieve a biotechnological production of indole will be based on the principle of design-build-test-learn-cycle inherent to systems and synthetic biology approaches. A metabolic model, describing all biochemical pathways of C. glutamicum, will be used to design a bacterium that can produce indole in an optimal way. The design will be tuned by using observations on the bacterium as a system, meaning that regulatory bottlenecks and unintended side products will be eliminated. For this purpose, we will design regulatory circuits that guarantee optimal flow of the metabolism to indole. The best indole-producing strain developed in this way will be tested in an industrial setting, to produce flavour-grade indole.

More broadly, the computational models and biosynthetic and regulatory building bricks generated in INDIE will be recruited to build a systems and synthetic biology framework for corynebacteria that will be easily extendable to new food ingredients derived from aromatic amino acids, thereby strengthening the potential of these bacteria for sustainable aromatic compound production. INDIE will accelerate technology transfer to the European level, hence opening new markets and strengthening European efforts to achieve sustainable industrial development.

Electric plug adapters for proteins: Activating iron-sulfur enzymes to fully exploit Nature's catalytic potential for biotechnology

PROJECT PARTNERS:

Contact Name of Organization Country
Gregory Bokinsky (coordinator)
Contact: g.e.bokinsky[at]tudelft.nl
Delft Technical University Netherlands
Frédéric Barras CNRS France
Miguel Alcalde Instituto de Catalisis y Petroleoquimica (CSIC) Spain
Georg Lentzen Isobionics BV Netherlands
Sandrine Ollagnier-de Choudens CNRS France

Project summary

Microbes are routinely engineered to synthesise chemicals from renewable materials. Improvements in microbial engineering are accelerating the global transition to a sustainable bio-based economy, as more chemicals are made in a manner similar to beer brewing, rather than being refined from oil. At the heart of microbial synthesis are enzymes that catalyse the reactions that produce the chemicals. Harnessing enzymes can be straightforward: genes encoding enzymes that perform the needed chemistry are installed into a microbial species, which then produces the chemical.

However, while cells readily manufacture enzymes from other species, often the enzymes cannot perform the needed chemistry. This prevents microbial engineers from using such enzymes, and as a result, many chemicals still cannot be made by microbes on a useful scale. This problem is severe for enzymes known as iron-sulfur (FeS) enzymes. These enzymes must obtain iron (in the form of FeS clusters) and electrons from their host cell, just like many devices need batteries and electricity. If we can better equip FeS enzymes with FeS clusters, we will improve their activity, making it possible to produce even more chemicals with microbes and further push the development of a bio-based economy.

In cells, iron and electrons are supplied to FeS enzymes by proteins that act as distribution networks. If the FeS enzymes are abundant (i.e. when overexpressed as part of an engineered pathway), these networks lack the capacity to deliver FeS clusters and electrons to FeS enzymes, and the FeS enzymes function poorly. Even worse, FeS enzymes taken from foreign species cannot “plug in” to the cellular networks. Using FeS enzymes to make chemicals therefore requires either an upgrade in the distribution network capacity to better supply FeS clusters and electrons, or adapters that enable the enzymes to plug in to the cell’s grid (just like electrical devices need adapters when traveling abroad). We find that foreign FeS enzymes can be better activated if parts of the distribution network from the foreign species (A-type FeS carriers, or “plug adapters”) are also abundantly supplied.

Inspired by our success, we will use improved FeS cluster and electron distribution networks to improve the activity of an FeS enzyme (IspG) that can be harnessed to make a family of compounds (isoprenoids), which includes biofuels, solvents, and fragrances. IspG is notorious for its poor activity, and research suggests this is due to an insufficient supply of FeS clusters and electrons. As a result, microbial engineers often rely upon a less-efficient isoprenoid synthesis pathway that avoids IspG entirely. Our Consortium has identified the A-type carriers that activate IspG enzymes with FeS clusters, and it is further known that supplying more electrons to IspG improves its function. We will improve the cellular distribution networks linked to IspG to help it produce isoprenoids more efficiently. We will then move our tests from the lab bench to an industrial setting by improving an efficient IspG-dependent fermentation process that manufactures fragrances and flavours from sustainable sources.

We will also search genomes for plug adapters to activate additional types of FeS enzymes. Activating FeS enzymes beyond IspG will demonstrate how to activate unique FeS enzymes, such as those needed to produce novel antibiotics. Such an approach will vastly expand the chemical versatility of engineered microbes.

Our Consortium will also design and evolve proteins that deliver FeS clusters to a broader range of FeS enzymes. Such proteins might act like universal power adapters, which could relieve the need to find a specific plug adapter for each FeS enzyme. We will also study how and why FeS enzymes and power grid components interact, or fail to interact. This will help us to better use FeS delivery systems, and further improve the performance of FeS enzymes.

MEmbrane Modulation for BiopRocess enhANcEment

PROJECT PARTNERS:

Contact Name of Organization Country
Alan Goddard (coordinator)
Contact: a.goddard[at]aston.ac.uk
Aston University (Aston) United Kingdom
Gavin Thomas University of York (York) United Kingdom
Amparo Querol Consejo Superior de Investigaciones Científicas (CSIC) Spain
Stephan Noack Forschungszentrum Jülich (FZJ) Germany
Siewert-Jan Marrink The University of Groningen (Groningen) Netherlands
Mustafa Turker Pakmaya (Pakmaya) Turkey
Jose Heras Lallemand Spain

Project summary

The global economy has an unsustainable dependence on fossil raw material with demand for raw material inputs to industry growing steadily. Concerns about environmental sustainability are becoming more acute; thus, alternatives to traditional, fossil-fuel based chemical production are urgently required. Cell factories, which use microorganisms to produce materials from renewable biomass, are an attractive alternative, and an increasing number of platform chemicals are being produced at industrial scale using engineered microorganisms. These are expected to have a transformative impact in industrial biotechnology, but, first, we must meet the challenges of designing and optimizing high-yield cell factory strains that can produce commercially viable amounts of product. One reason for poor product output is that the production conditions are ultimately toxic to the producing cells. In addition to damage to intracellular components such as enzymes, the lipid cell membrane and associated proteins are vulnerable to biomolecules e.g. ethanol and propionate, as well as to physical parameters during production such as osmotic stress, pH, and temperature. An approach whereby membranes can be “tuned”, in terms of their lipid and protein content, to become more resistant to stresses brought about by toxicity would revolutionise the field. Additionally, expression of efficient membrane transporters to export ‘toxic’ products can mitigate intracellular damage. These approaches will ultimately enable production of higher concentrations of the desired molecules or cells making the bioprocesses more efficient, increasing product yield, reducing cost, and help to drive the move away from fossil-based raw materials. An adoption of such “green” processes and avoidance of depletion of non-renewable carbon sources will bring huge social and environmental benefits. Products and processes which are currently economically unviable due to toxicity can be rendered profitable by even small increases in the resistance of strains and concomitant yield increases.

This 36-month project sees five leading research institutes (Aston, York, FZJ, IATA-CSIC and Groningen) and two large industry partners (Lallemand and Pakmaya), across five countries, collaborate, and validate at pilot scale, engineered robust cell factories (yeast and Propionibacterium) that overcome existing toxicity challenges, improve efficiency and allow their effective commercialisation. The strategies developed within this project will be applicable across the sector to facilitate rational strain engineering with far-reaching benefits.

The project is divided into seven interconnected, iterative work packages (WPs) with a well-established build-test-analyse approach. Initial analysis of –omics data will identify key alterations in membrane protein and lipid content of both microbes subjected to stresses associated with bioproduction and those strains known to be somewhat resistant to such stresses (WP1). In vitro and in silico approaches will be used to rapidly delineate the roles of these alterations and rationally design more resistant membranes (WP2). Using synthetic biology and strain evolution approaches, we will alter the membrane composition of microbes to reflect the “optimal” membranes determined in WP2 (WP3). Optimal strains will be identified in a high throughput manner and subjected to large-scale testing to ensure that the changes made translate to the industrial setting (WP4). Following this, another iteration of the cycle will further optimise the strains. WP5 will evaluate the environmental and social sustainability of the innovative production processes and the final products. WP6 will develop and implement a strategy for the dissemination and exploitation of research results to different stakeholders. WP7 involves consortium management, project governance, communication activities and administrative oversight to ensure maximum impact of the project.

MicroalgaE as Renewable Innovative green cell facTories

PROJECT PARTNERS:

Contact Name of Organization Country
Olaf Kruse (coordinator)
Contact: olaf.kruse[at]uni-bielefeld.de
Bielefeld University Germany
Alison Smith University of Cambridge United Kingdom
Rene Wijffels Wageningen University Netherlands
Josue Heinrich Universidad Nacional del Litoral Argentina
Andrew Spicer Spicer Consulting Limited United Kingdom

Project summary

This project [MERIT] will leverage state of the art synthetic biology techniques to engineer microalgae for the sustainable production of high-value, medically and industrially relevant novel diterpenoid products from carbon dioxide and light. Twenty carbon containing (C20) diterpenes are complex, often oxy-functionalized secondary metabolites found largely in plants. Their variety and complexity have made them incredibly interesting for numerous applications as medicines, antimicrobial agents, and high-value chemicals. The complex structures of diterpenoids are difficult and costly to chemically synthesize and can be expensive or inefficient to purify from their native host organisms. All organisms produce the same 5-carbon building blocks used in terpenoid production pathways. Heterologous expression of modular terepene synthase pathways can be used to produce non-native terpenoids in engineered hosts. Production of terpenoid products in fermentative hosts has become a mature technology, however, relies on unsustainable use of organic carbon sources such as glucose and inherently competes for agricultural resources. Fermentative microbial hosts are additionally not optimized for the production of the C20 diterpenoid precursor geranylgeranyl pyrophosphate (GGPP). Microalgae, however, are naturally optimized to produce GGPP as the precursor for light harvesting and photoprotective pigments in the cell. Over-expression of diterpene synthases (DiTPS) allows the conversion of this precursor into the numerous carbon skeletons of diterpenoid products. Algal cells also represent ideal chassis for the expression of cytochrome P450 monooxygenases (CYPs), which are needed for oxy-functionalization of diterpene backbones to specialized structures. In photosynthetic cells, CYPs can be coupled to photosynthetic electron transport chains for efficient redox potential, an otherwise limiting factor in non-photosynthetic hosts. Combination of DiTPS and CYPs in an algal chloroplast is the ideal chassis for complex oxy-functionalized diterpenoid production, where both high concentrations of precursor and the redox electron flow are abundant. Algae hold the additional benefit of rapid growth rates in simple mineral salt solutions using only light and CO2 as energy inputs.

These organisms are ideal hosts for the production of diterpenes and are inherently sustainable production chassis. To date, engineering of microalgae for robust expression of transgenes has been a major limiting factor to their widespread application as green-cell factories for complex biotechnological targets. Through combined efforts of strain domestication and synthetic biology, the development of synthetic eukaryotic algal transgenes (SEATs) have recently been demonstrated to facilitate advanced engineering of these highly promising organisms.

In this project, an international team of experts in algal synthetic biology, cultivation, and industrial process modelling, will join forces to design optimized algal strains and the accompanying industrial production as well as product extraction processes for light-driven conversion of CO2 to high-value diterpenoid products. This collaborative team represents global leaders in algal synthetic biology, outdooralgal cultivation, photobioreactor design, and process modelling. The MERIT team already successfully engineered pathways for the production of several diterpenoids in microalgae. Multiple levels of strain engineering and synthetic biology will be implemented to create green-cell factories with enhanced carbon flow from CO2 to terpenoids. Various DiTPS and CYPs will be combined to produce novel ‘new-to-nature’ diterpenoid products with incredible potential for numerous applications. Optimized strains will be grown to scale and processes for diterpene product extraction designed. The project will generate many new avenues of commercialization potential and significantly contribute to the development of the European Bioeconomy.

Streamlined Streptomyces cell factories for industrial production of valuable natural products

PROJECT PARTNERS:

Contact Name of Organization Country
Andriy Luzhetskyy (coordinator)
Contact: a.luzhetskyy[at]mx.uni-saarland.de
University of Saarland Germany
Rolf Müller Helmholtz- Zentrum für Infektionsforschung HZI/HIPS Germany
Hrvoje Petkovic University of Ljubljana Slovenia
Francisco Moris ENTRECHEM Spain
Gregor Kopitar Novartis (Lek pharmaceuticals d.d.) Slovenia

Project summary

Natural products cover a unique chemical space, which is particularly well-suited for the development of antibiotics and anticancer drugs. Actinomycetes are the richest source of natural products such as antibiotics for medical, veterinary and agricultural use, and therefore represent a class of bacteria of considerable interest for the human welfare. Indeed, there are huge number of novel compounds discovered so far, however, the supply of sufficient amounts for studying of pharmacological properties is a significant bottleneck in drug development. Our project will create a robust natural products supply platform based on the powerful industrial oxytetracycline overproducer Streptomyces rimosus. The concept integrates systems and synthetic biology with bioinformatics and process engineering into a purpose-driven and engineering workflow. Multi-omics analysis of this strain will deliver key insights for targeted optimization into superior chassis. During development, the iterative and interactive combination of carefully tailored experimental and computer-modelling approaches will support the prediction of multi-combinatorial genetic traits to develop a superior microbial chassis. A full range of new synthetic parts, such as fine-tuned promoters, terminators and regulatory circuits as well as cutting-edge CRISPR/Cas9 genetic engineering will be developed for an exact, marker-less and fast translation of identified, desired features into a clear genetic language, operated by the created S. rimosus cells. In addition to biosynthetic power, project will consider cellular genetic stability, process tolerance and robustness by pre-early integration of expected needs from industrial partners into the design process. The project will enable a tailor-optimised production of valuable bioactive compounds for downstream development as pharmaceuticals.

Biovalorization Of Olive Mill Wastewater To Microbial Lipids And Other Products via Rhodotorula Glutinis Fermentation

PROJECT PARTNERS:

Contact Name of Organization Country
Alper Karakaya (coordinator)
Contact: alper.karakaya[at]gmail.com
Düzen Biological Sciences R&D and Production Company (DUZEN) Turkey
Blaž Likozar National Institute of Chemistry Slovenia
Marta Benito Asociación para la Investigación, Desarrollo e Innovación del sector Agroalimentario (AIDISA) Spain
Gaetano Perrotta The Agenzia nazionale per le nuove tecnologie, l'energia e lo sviluppo economico sostenibile (ENEA) Italy
Daniel Pleissner Leuphana University of Lüneburg Germany
Friedrich Herberg University of Kassel (UniKassel) Germany
Egils Stalidzans University of Latvia Latvia

Project summary

Olive mill wastewater (OMW) is a significant by-product of the food industry of the olive oil producer countries in Mediterranean basin, with a high environmental impact, when not appropriately treated. However, at the same time OMW is rich in organic compounds, which can either be used directly after extraction, or valorized via biocatalytic processes. RHODOLIVE suggests an innovative circular bioeconomy approach for the valorization of this side-stream of the food industry, by treating the OMW with a non-conventional yeast, in order to accumulate and produce microbial lipids, biophenols and carotenoids, which will be used in the development of functional food products.

To be able to fulfill the goals of RHODOLIVE, a multidisciplinary approach is adopted, that harmonically utilizes the know-how of different fields. Six different lands are participating in our consortium, several of them already facing the challenge of the treatment of OMW, as olive oil producing countries. The partners participating have complementary expertise so that a holistic approach to the valorization of OMW is possible. More specifically, DÜZEN Biological Sciences Research and Development and Production Co. (DÜZEN, Turkey) will provide the know-how of the fermentation of R. glutinis that accumulated since 2009, in their collaboration with Ankara University. A pilot scale bioreactor (30 L) will be installed near the Laleli olive oil company (owned by DÜZEN) in Balikesir, to perform the preparative fermentation and the optimisation studies. The studies will focus on the yeast biomass and several bioproducts of the fermentation, namely lipids, biophenols (such as luteolin) and carotenoids (β-carotene). The National Institute of Chemistry in Slovenia will contribute their expertise in the development of “green” extraction systems for the purification of the aforementioned products from the delivered biomass. University of Kassel (UniKassel, Germany) will use enzymes to transform the isolated phenolics, to improve their organoleptic properties. The final isolated biocompounds and the biomass will be used from Asociación para la Investigación, Desarrollo e Innovación del sector Agroalimentario (AIDISA, Spain), which will perform studies on their implementation in food products. In parallel, to minimize the environment impact even further, Agenzia nazionale per le nuove tecnologie, l'energia e lo sviluppo economico sostenibile (ENEA, Italy) in collaboration with Leuphana University of Lüneburg (Germany) will identify fungi and algae that can further degrade the effluent of the fermentation, while via proteomic and metabolomic studies novel nutraceutical compounds will be identified from these organisms, that will increase the impact of the developed circular bioprocess. In parallel to this chain of delivery, University of Latvia will provide an insight on the metabolic pathways of R. glutinis with their expertise on metabolism modeling. This study will provide constructive feedback to improve both the fermentation, by identifying the bottlenecks and trying to surpass them with changing the fermentation parameters, and the strain, by producing (in collaboration with UniKassel) novel improved strains of R. glutinis that could later be used to further increase the titer, to establish a sustainable bioprocess.

Though this integrated treatment of OMW we envision the sustainable and environmentally friendly bioprocess that will provide a competitive niche in food industry. RHODOLIVE will be performed in industrial related environment, using pilot-scale bioreactor, while all the subsequent steps will be in preparative scale. Aim of RHODOLIVE is to develop a bioprocess for providing industrial synergy and contributing to a circular bio-economy and reducing the carbon footprint.

Investigating large scale bioreactor effects in microbial application

PROJECT PARTNERS:

Contact Name of Organization Country
Marco Oldiges (coordinator)
Contact: m.oldiges[at]fz-juelich.de
Forschungszentrum Jülich Germany
Jacques Georis Puratos NV/SA Belgium
Michael Ferguson Gymetrics France SAS France
Stefan Junne Technische Universität Berlin Germany
Raivo Vilu Center of Food and Fermentation Technologies Estonia

Project summary

The biotechnological production of enzymes and chemicals for industrial purposes in large scale bioreactors is state-of-the-art technology. However, environmental conditions are no longer homogenous in production scale, due to the formation of gradients in different zones of the bioreactor vessel (i.e. substrate, oxygen, pH, T, pCO2). Such gradients are often responsible for performance losses during scale up. Although this is known for a long time, still no consistent strategy exists how to precisely scale down, tailored to the bioreactor and process. Rough estimations rather than knowledge-based approaches are typical, which prevent cost-efficiency during scale up. Not much advantage is taken from efforts made in sensor, -omics and strain engineering technology and computational approaches to directly address an increase of strain and process robustness related to large scale issues. Nevertheless, problems related to scale up are among the most important factors that reduce investments for the implementation of bioprocesses for novel products after a host strain has been identified at lab scale.

The ScaleApp project address the next generation design of scale-down bioreactors by smart combination of powerful CFD with innovative process analytical technology (PAT). CFD will be applied to model different bioreactor geometries from pilot-scale and production scale bioreactors. The CFD models will be validated using PAT allowing real-time measurements inside the bioreactor during the cultivation process. This is enabled by tailor-made multi-position sensor devices already in place and functional, which can be moved along the height axis inside pilot and production scale. Thus, the multi-position sensor devices precisely track concentration gradient profiles along the bioreactor height. In order to shed light on gradient distribution in the full space of the bioreactor volume, wireless free flowing small sensor devices of approx. 25 mm diameter will be applied to enable real-time dynamic measurements.

For the first time, this will enable substantial validation of CFD and hybrid models with comprehensive data sets of spatially and timely resolved data. Validated CFD models will be used to optimise the design of a corresponding scale-down bioreactor setup. This tailor-made scale-down bioreactor setup will be physically built, validated and compared to its pilot scale representation. Different scale-down bioreactor setups will be validated for their potential and standards will be set to achieve applicable and precise scale-down applications.

ScaleApp also addresses metabolic analysis of the selected microbial systems under these precise scale-down conditions. Such investigation will deal with industrially relevant yeast strain as model for eucaryotes and protein secreting Bacillus strains as model for procaryotes with industrial partners. Stress response to gradients will be elucidated, i.e. at varying supply of substrate, oxygen, T or pH gradients. This is performed on the level of comparative -omics data sets, i.e. transcriptomics, proteomics, metabolomics, single-cell based physiologic and morphologic analysis. Metabolic targets identified are used to genetically engineer the microbial hosts for increased metabolic robustness providing superior applicability in large scale production. Models will be used to define acceptable operation ranges in order to describe opportunities for improved engineering of the large scale.

Improvements in the CFD-assisted design of scale-down bioreactor models and their application at early-stages of strain engineering and bioprocess development, will allow critical decisions at the level of the lab-scale rather than at the level of production scale. From an economic point of view this reduces risk of failure and more reliable estimation of product development times and time-to-market. The economic impact of ScaleApp will be evaluated by process cost analysis by the industrial partners and LCA.

Sustainable Bioproduction of Pheromones for Insect Pest Control in Agriculture

PROJECT PARTNERS:

Contact Name of Organization Country
Diego Orzaez (coordinator)
Contact: dorzaez[at]ibmcp.upv.es
Agencia Estatal Consejo Superior de Investigaciones Científicas Spain
Nicola Patron Earlham Institute United Kingdom
Heribert Warzecha Technische Universität Darmstadt Germany
Spela Baebler National Institute of Biology Slovenia
Teresa Gadea ECOLOGIA Y PROTECCION AGRICOLA SL Spain

Project summary

The aim of this project is to enable bio-based manufacturing of insect pheromones in plants and fungi for the sustainable control of insect pests of agriculture and horticulture. This builds on a proof-of-concept in which moth sex-pheromones were produced in plants.

Some of the most aggressive pests of agriculture are insect larvae. Semiochemicals are chemicals emitted by insects for communication. The most widely known of these are sex pheromones, produced by virgin females to attract mates of the same species. Dispensing insect sex pheromones in plant production environments is used to trigger sexual confusion in the target species and prevent breeding, thereby providing a highly species-specific control method. This presents a sustainable alternative to conventional pesticides, the use of which are progressively being restricted due to concerns about their non-specificity and negative impacts on biodiversity.

Insect sex pheromones are already used as a pest-control strategy; however, chemical synthesis is currently the only approach for manufacturing and the use of toxic ingredients and the creation of toxic by-products is inevitable for some pathways. Further, the unusual chemical characteristics of many insect pheromones mean that chemical synthesis is not cost effective. For example, Coccoidea species (scale insects and mealybugs) are aggressive pests of agriculture and horticulture and better control methods are highly desirable. However, their sex pheromones have unusual irregular terpenoid structures for which chemical synthesis is both difficult and expensive.

The SUSPHIRE project aims for the bioproduction of insect sex pheromones in plants and fungi. Previous studies by SUSPHIRE partners demonstrated that it is possible to engineer N. benthamiana plants to produce high quantities of moth sex pheromones via heterologous expression of the insect enzymes. The SUSPHIRE project will improve on this initial proof-of-concept and will also identify and validate key biosynthetic enzymes for the bioproduction of Coccoidea pheromones. We will take a synthetic biology approach, informed by data obtained from bioinformatics and systems biology analyses, to identify and refactor insect pheromone biosynthesis pathways, optimizing bioproduction in plants and fungi.

The SUSPHIRE project will demonstrate that biosynthesis can provide a sustainable, low-cost manufacturing platform for the commercial production of insect pheromones and reduce the cost of production of pheromones that are currently commercially non-viable. The long term aim is production of a living bio-dispenser but SUSPHIRE will produce several intermediate marketable products including pheromone-enriched biomass; bioproduced precursors that can be used to bypass unfavourable steps and reduce the cost of chemical synthesis; and enzymes to assist chemical synthesis of complex precursors. The introduction of these biotechnology approaches to pheromone production will expand the use of sex pheromones for sustainable pest control in agriculture, reducing its current environmental impact and providing sustainable manufacturing platforms.

Tobacco as sustainable production platform of the natural biopolymer cyanophycin as co-product to oil and protein

PROJECT PARTNERS:

Contact Name of Organization Country
Carlos Dezar (coordinator)
Contact: carlos.dezar[at]indear.com
BIOCERES S.A. Argentina
Inge Broer University of Rostock Germany
Jeroen Hugenholtz Wageningen Research Netherlands
Samantha Hampton Idroedil SRL Italy
Justus Wesseler Wageningen UR Netherlands
Ursula Weisenfeld Leuphana University of Lüneburg Germany

Project summary

This project combines plant and industrial biotechnology to increase the value of commercially grown tobacco with products that can sustainably substitute fossil raw materials. We aim to establish an economically feasible production system for the biopolymer cyanophycin (CGP) as a byproduct without relevant additional costs, which can be adopted by farmers and biotech companies in Argentina.

CGP is a biopolymer of β-Asp-Arg dipeptides, which is produced naturally by cyanobacteria and can be used as both a valuable N-rich ingredient for the feed industry and as a novel biopolymer for the chemical and material industry. The heterologous production of CGP has already been established in bacteria, yeast and tobacco plants with yields up to 40%, 21% and 9.4% dry weight, respectively. None of these were translated into an industrial production system or brought to market application.

This project aims at achieving its goals with a consortium of five expert research centers and industries in the area of tobacco and CGP, namely Bioceres (Argentina), University of Rostock and the Leuphana University (Germany), Wageningen University and Research Centre (Netherlands) and Idroedil (Italy). The project tasks are organized in three Workpackages (WP’s) focusing on 1. Optimization of CGP production by existing CGP-producing tobacco varieties, efficient CGP isolation from tobacco and development of CGP application, 2. Introduction of CGP-production in the Solaris tobacco bred for high oil content, implementation of resulting CGP-tobacco lead events in tobacco-growing sites in Argentina and development of CGP, oil and protein isolation protocols for a pilot plant and 3. Social and Economic impacts of the novel CGP-producing tobacco cultivars.

In WP1, CGP-production by modified Virgin and Burley tobacco will be optimized on the field, focusing on leaves and seeds as source. For optimal extraction of CGP, a biorefinery concept will be developed based on the Grassa technology developed for extraction of proteins and other nutrients. The isolated CGP can be used either as ingredient/N-source for the feed industry, as a biopolymer as such, as a basis for novel biopolymers after chemical conversion to diaminobutane, and/or for production of polyaspartate after mild hydrolysis (to polyaspartate and arginine). These novel biopolymers will be tested alone, in blends/mixtures or in combination with other biopolymers such as starch, cellulose and lignin, some of which are also components of the tobacco plant.

In WP2, CGP production will be introduced in the Solaris tobacco. This variety has been developed for its ability to grow under marginal conditions and for high oil- and no nicotine content. This tobacco oil is a potential, local, source for biofuel. By combining the production of biodiesel, protein, fiber and CGP, the economics for this process should become more favorable.

Finally, in WP3, the economics and social impact of CGP-production by the different tobacco varieties will be assessed. A socio-economic, partial equilibrium, model will be developed to predict the effect of CGP-production, farming, processing, retailing, etc. in Argentina and in the partner countries. A Technical Economic Evaluation and Life Cycle Analysis will be conducted for the process of CGP-production by tobacco. For this, the production costs for CGP by tobacco will also be compared with other existing CGP-producers such as the Cyanobacteria, E.coli or the modified yeast (Saccharomyces cerevisiae).

With this approach, the consortium expects to develop strong alternatives for the tobacco industry, in general, and especially to geographical areas such as (Northern) Argentina that rely strongly on the growth of this crop.

The processes and outcomes of the project will be aligned with socio-economic issues of the countries involved, thereby providing knowledge for implementation.

Synthetic Biology for Industrial Production of Steroids

PROJECT PARTNERS:

Contact Name of Organization Country
Alberto Sola (coordinator)
Contact: alberto.sola[at]inbiotec.com
Asociación de Investigación (INBIOTEC) Instituto de Biotecnología de León Spain
Marina Donova Pharmins Ltd Russia
Havard Sletta Sintef Materials and Chemistry Norway
Gerhard Schembecker TU Dortmund Germany
José Luis Barredo Bionice Spain

Project summary

Steroids are the second largest class of drugs marketed for medical applications, only exceeded by antibiotics. Currently, there are more than 300 clinically approved steroidal compounds, representing a 10 billion USD market. Although the production of key steroid intermediates (mainly C19-steroids) from phytosterols has been industrially implemented, there is still considerable room for improvement and many challenges remain. The generation of effective engineered strains, improved bioprocess efficiency, improved product recovery, and the problem of end-product inhibition are major challenges for the industry. The Syntheroids (Synthetic Biology for Industrial Production of Steroids) project aims to develop integrative processes for innovative bioconversion of phytosterols to C22-steroids based on Synthetic Biology of non-pathogenic Actinobacteria (Mycobacterium, Nocardioides). These compounds are key intermediates in the synthesis of several therapeutic steroids applied in gastroenterology and endocrinology.

The central objective of Syntheroids is to develop an integrated production process for pharmaceutical steroids using Synthetic Biology and improved processing technology. To achieve this goal, the Syntheroids project has the following four specific objectives:

  • Omics data integration from steroid producing Actinobacteria as a source of Synthetic Biology targets for productive strain evolution
  • Creating genetically engineered bacterial strains capable of producing innovative C22‑steroid precursors
  • Reduce or eliminate end-product inhibition by mutagenesis, genetic engineering and process optimization.
  • Integrate up- and downstream processes for an eco-friendly bioconversion.

Steroid pharmaceuticals are important for life quality, healthy development and ageing, all major challenges today. Production of key intermediates for the synthesis of therapeutic steroids, in an eco-friendly and economical process, is the main expected result of Syntheroids. A few steroid precursors are today industrially produced from phytosterols, mainly by companies in China, India and the US, although some are located in Europe. All in fierce and keen competition. Innovative ideas that can expand the list of steroids produced from phytosterols in a single-step biotechnological process are wanted, and this is why two European companies (Bionice and Pharmins ltd.) are active partners in the five-partner Consortium Syntheroids. Shorter steroid production pipelines and eco-friendly processes, in compliance with European regulations, will increase the EU-GDP (gross domestic product) as it will increase the companies’ competitiveness and reduce the end drug user's medical invoice.

Enzyme platform for the synthesis of chiral aminoalcohols

PROJECT PARTNERS:

Contact Name of Organization Country
Wolf Dieter Fessner (coordinator)
Contact: theodoridou[at]mail.oc.chemie.tu-darmstadt.de
Technische Universität Darmstadt Germany
Pere Clapés Consejo Superior de Investigaciones Científicas - CSIC - Institute of Advanced Chemistry of Catalonia - IAQC Spain
Laurence Hecquet Université Clermont Auvergne (UCA), CNRS, SIGMA Clermont France
John Ward University College London United Kingdom
Simon Charnock PROZOMIX United Kingdom
Michael Breuer Germany LE BASF SE Germany

Project summary

Amino alcohol moieties are found in highly diverse classes of natural products that are of great importance due to their bioactivity, and they function as chiral building blocks for the synthesis of pharmaceuticals and agrochemicals. The chemical synthesis of stereoisomerically pure amino alcohols is difficult and typically requires uneconomical steps for protective group manipulations. Conventional syntheses in the chemical industry often use hazardous substances, consume large amounts of energy and generate toxic waste.

The TRALAMINOL project will address the challenges with the innovative development of sustainable biotechnological processes for the synthesis of amino alcohols through a multi-disciplinary approach.The consortium assembles leading European research groups (Germany, UK, Spain, France) with different but complementary scientific and technological expertise (4 non-profit organizations, 1 SME, 1 large company). TRALAMINOL will focus on the development of a powerful one-pot two-step biocatalytic strategy based on only two classes of reaction types: The approach makes use of key enzymes that catalyze C–C bond formation followed by enzymatic amino transfer in highly controlled fashion by exploiting the enzymes’ high chemo-, regio- and enantioselectivity, while operating under mild reaction conditions. The project will span TRL 3-7 by incorporating demonstration reactions at technical scale (up to 10L reactor volume) carried out by industrial
partners.

The reaction cascades are designed so that the choice of substrate and specificity of the enzymes involved will generate different target structures in a controlled fashion. At an advanced stage this technology may be transferred into recombinant whole-cell catalysts to improve overall economics for industrial applications. Substrates are bio-based, sustainable building blocks that will be transformed into molecules with high added value. Compounds with low water solubility can be addressed by the use of solvent stable enzymes from thermophiles. The consortium has identified a series of relevant target structures from different compound classes that are important representatives for diverse billion-€ pharmaceutical market segments, including drugs related to treatment of cancer, circulatory disease, diabetes, microbial infection and others. With its one third industrial participation and top-down approach to identified market needs, the consortium not only represents academic excellence but also covers commercial interests at different stages of the value chain. Thus, the processes developed within the TRALAMINOL project and evaluated against their economic viability can achieve a significant environmental impact by replacing more energy and resource intensive processes, leading to reduced environmental footprints and lowering our dependence on fossil raw materials.

Enzyme portfolios of each reaction type designed for maximum genetic diversity will be developed as a robust industrial biocatalytic platform. Such superfamily panels will be manufactured by a unique technology platform for massive recombinant protein expression and production, enabling gram quantities of 1000s of enzymes. Such carefully designed enzyme selection panels significantly reduce the time and effort required to identify the best catalyst for novel target substrates. Further intense development of these two critical enzyme types, and the creation of an integrated reaction platform for the sustainable manufacture of multifunctional chiral building blocks, will significantly strengthen the global competitiveness of the European chemical and pharmaceutical industries and accelerate the transition from a dependence on fossil raw materials toward a sustainable bio-based economy.

Environmentally-friendly bioadhesives from renewable resources

PROJECT PARTNERS:

Contact Name of Organization Country
María Teresa Moreira (coordinator)
Contact: maite.moreira[at]usc.es
University of Santiago de Compostela Spain
Antonio Pizzi Universite de Lorraine France
Milan Šernek University of Ljubljana Slovenia
Marie Pierre Laborie Albert Ludwig University of Freiburg Germany
Detlef Schmiedl Fraunhofer ICT Germany

Project summary

Wood adhesives are of tremendous industrial importance as more than two-thirds of wood products in the world are totally, or at least partially, bonded together using a variety of adhesives. Synthetic adhesives greatly dominate the field of binders for wood and fiber panels, their annual world production running in the million of tons. Formaldehyde is a key building block in formaldehyde-based glues and resins used for the production of wood-based panels, which constitute more than 50% by volume of all the adhesives used today. The acceptable levels of formaldehyde emission from wood panel products have been continuously reduced over the last decades. The driving forces have been the increased public awareness and the consumer demand for non-hazardous products as well as the corresponding governmental regulations. Bio-based polymers such as proteins, tannins, lignins and carbohydrates are today being explored as possible adhesives due to their relative abundance and promising properties. Despite the intense research in this field, obtaining a natural free-formaldehyde formulation is still a challenge.

This project aims to study the feasibility of replacing formaldehyde in wood adhesives by natural components derived from wood or other vegetable matter. The consortium will develop new bioadhesives which are able to provide a holistic solution to the current emissions challenges facing the wood-based composites industry. The proposed solution is focused on different modifications of polyphenols, namely lignin and tannins, for producing bioadhesives that do not contain formaldehyde in its formulation, eliminating in this way the emissions of volatile organic compounds (VOC). The substrates will be Kraft lignin, from the pulp and paper industry, hardwood Organosolv lignin, as well as mimosa, quebracho and chesnut tannins. Depending on the nature of the raw material, some substrates may need to be modified at different levels for increasing their reactivity. Chemical and enzymatic approaches will be applied to modify the substrates. Then, on the modified and unmodified materials novel reactions to produce the bioadhesives will be evaluated. The key aspect to obtain such bioadhesives is the system of hardening, without which any modification will be of no use.

The consortium of the project is formed by five partners from four countries (France, Germany, Slovenia and Spain). The topic of the project is relevant to the economy of these countries. Germany is the leading country in the production of wood panels in the EU, being followed by other countries such as Poland and France. The EU wood panel industry has an annual turnover of about 22 billion euros, creates over 100000 jobs directly and counts with more than 5 000 enterprises in Europe.

The use residual by-products (lignins, tannins) from important European industries (e.g. cellulose producers, 2G biorefineries) for producing a compound that is intended to replace a chemical with a high toxic and environmental load in the wood-composite industry foresees the application of circular economy, expanding the concept of biorefinery. The valorization of residual/by-products streams into bioadhesives, that will be used to reduce the release levels of VOC, will have a significant impact on the wood-based composite industry. Besides, different sectors of society ranging from wood-based composite industry, workers and people living around industrial plants using formaldehyde in their process, and the society in general, will benefit from the results of this project. The information and data obtained along the project will be managed and published taking into account the guidelines of the European Research Council, as stated in the implementation of Open Access to Scientific Publications and Research Data and FAIR Data Management, respectively.

Microbial community modeling for the production of ‘designer’ yogurt

PROJECT PARTNERS:

Contact Name of Organization Country
Bas Teusink (coordinator)
Contact: b.teusink[at]vu.nl
Vrije Universiteit Amsterdam Netherlands
Ursula Kummer Heidelberg University Germany
Ralf Takors University of Stuttgart Germany
Petri-Jaan Lahtvee University of Tartu Estonia
Ana Rute Neves Chr. Hansen A/S other (Denmark)

Project summary

Many industrial biotechnological processes are carried out by consortia of bacteria, rather than single strains. To improve performances of such processes, the biotech industry currently relies mostly on screening-based selection of isolated strains with desired properties. However, these properties are very often influenced by other consortium members in unknown ways. Screening consortia is challenging, because only a tiny subset of all the many possible combinations can ever be tested. There is therefore a need to develop methods that can predict performance of strains in consortia, on the basis of the genome and selected phenotypic traits. This project aims to develop an integrative bioinformatics and modeling approach to predict microbial community functioning from the properties of the constituent isolates. We will do this through a real industrial use case: the design of microbial cultures for the production of yogurt.

Industrial-scale yogurt production is carried out with a broad range of cultures consisting mainly of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus strains. Different cultures (a blend of typically 2 to 5 different strains) are formulated to obtain desirable characteristics in the final product, such as fast acidification to a desired acidity, optimal texture, reduced fat levels, proper sweetness or desired flavor profile. Understanding the genetic determinants of variability between strains related to such functionalities is a key question in the industry. However, the overall function of milk-fermenting strains can largely be modulated by the metabolic interactions between the individual strains. Yogurt fermentation therefore is an excellent test case to develop rational community designer methods, as it consists of relatively few species and its interactions and industrially-relevant properties are based on metabolism – an area amenable to rigorous experimental and computational analyses.

Our approach is twofold: A data-driven approach can identify which features (genes, sequence variations, metabolic network attributes, protein expression) predict functionality in the consortium. This requires data, which partly is available at Chr. Hansen at the start of the project, and partly will be collected in this project. This will include growth, as well as key parameters for functionality such as flavour and acidification profiles, for hundreds of combinations of strains in different media. The resultant statistical models can be directly used by Chr Hansen to prioritise combinations of strains based on features of the individual strains.

However, to explore the space of possibilities beyond what was screened for, mechanistic insight into community function will be required. Therefore, in parallel the project will develop mechanistic models that quantitatively and comprehensively describe the metabolic performance of representative S. thermophilus and L. bulgaricus strains alone and in co-culture, first in chemically defined media, later in milk. Knowledge on strain interactions will be learned from state-of-the-art omics data acquisition in combination with a suite of (genome-scale) modeling approaches. Through structural and parameter sensitivity analysis we will subsequently identify the key interactions and parameters that determine community function, and link those to strain diversity and culture design. This should enable Chr Hansen to explore the full potential of various S. thermophilus - L. bulgaricus combinations for the targeted production of yogurt with desirable properties, in this case acidity and flavour. In this way, this project sets an example how the improvement of industrial fermentations by microbial communities can be achieved through systems biology.

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