Final Report Summary - KYROBIO (The discovery, development and demonstration of biocatalysts for use in the industrial synthesis of chiral chemicals.)
Executive Summary:
The 4-year KYROBIO project aimed to improve the range of high value single enantiomer chiral chemicals manufactured in Europe using biotechnological routes. The use of industrial biotechnology for the production of chiral chemicals has already shown great potential. Effective competition with traditional chemo catalysts has been achieved in many instances, through specific targeted synthesis of the enantiomer of choice, thus increasing the total production yield of product. However biocatalysis methodologies still havegaps within its capabilities, when compared to the breadth of chemo-catalysis. The KYROBIO project is filling some of these gaps by developing new classes of enzyme catalysts and the associated process and economic knowledge needed for their exploitation.
The principle focus of research effort in KYROBIO was to enable enabling the industrial application of the lyase class of enzymes. Lyase enzymes have potential to expand the application of biocatalysis in chemical synthesis - they can selectively synthesize molecules with multiple chiral centres, based on the formation of carbon-carbon and carbon-nitrogen bonds. Increasing the availability and applicability of a variety of novel lyase enzymes suitable for industrial applications was a central objective of the KYROBIO project.
The development and application of these biocatalysts was supported by many technical disciplines working in concert. Technical approaches integrated enzyme discovery strategies; bioinformatics; structural studies; enzyme production platforms (fermentation, expression); enzyme process engineering; and biocatalyst formulation. This multidisciplinary approach was developed to expand and enhance the ranges of technical tools and skills needed to establish both the biocatalyst’s credentials and the supporting technologies to open exploitation routes for application of enzymes in the manufacture of high value chemical products.
A strong emphasis on dissemination from the project outset provided engagement with both specialist audiences and the wider public. A programme of networking events, publications and online communications, webinars, public engagement and specialised training events has been delivered. A major dissemination event held in Brussels to disseminate the project results and to promote the benefits of collaborative research in the development of Europe’s’ capability in Industrial Biotechnology. This was augmented by publication of original research in prestigious journals, and presentations at international conferences. Wider audiences were addressed in Science Open Days, Museum exhibitions, and networking events aimed at informing and engaging school children with biotechnology-themed topics.
The KYROBIO project has a 17 -member consortium comprising 5 Universities, 11 SMEs and a large multinational company. Complementary technical and commercial skills represented in the consortium have been a major project strength and have facilitated close collaborations to allow mutual exchange of fundamental knowledge, applied research techniques and technology development processes. KYROBIO’s largely industry focussed approach, coupled with strong SME participation and leadership, has been used to address industrially-identified needs for chiral synthesis using biocatalysis, and supply the necessary supporting technologies. Exploitable outputs include - novel biotransformations of interest to speciality chemical and pharmaceutical manufacturers; new technical platforms for enzyme discovery; bioinformatics/ structural based tools for enzyme improvements; expression and fermentation techniques to enhance biocatalyst manufacturing; and process and product recovery technologies to increase efficiency of biocatalysis processes. The strong industrial focus of the project has been successful in fully engaging participants who can exploit the added value of biocatalysis. At the project end date (30 Nov 2015) participants have taken some of the output from the KYROBIO project to market, and have advanced plans for further exploitation of the knowledge and materials developed within the project to generate new competitive products and services for the European chemical manufacturing industry.
Beneficiaries C-TECH INNOVATION LTD (Coordinator), evocatal GmbH , BiCT SRL, Ingenza LTD,CLEA TECHNOLOGIES BV, BIOINFOBANK INSTITUTE, PROZOMIX LIMITED, VTU TECHNOLOGY GMBH, x- ZYME GMBH,BIO-PRODICT BV, ACIB GMBH, UNIVERSITAET GRAZ UNIVERSITY OF MANCHESTER, RIJKSUNIVERSITEIT GRONINGEN, DANMARKS TEKNISKE UNIVERSITET, UNIVERSITAET STUTTGART, CI-KTN (now KTN LTD).
Project Context and Objectives:
The KYROBIO project was planned and delivered with the objective to generate a toolbox of new enzymes and technologies that could be used to synthesize single enantiomer chiral chemicals to be produced by industry in Europe using biocatalytic routes.
The main objective of KYROBIO was to enhance the competitiveness of the European chemical industry by substituting complex chemical syntheses using multi-step (often with precious metal catalysed reactions in hazardous chemical plants) by clean and non-polluting bioprocesses and to develop advanced engineering solutions and routes to market to enable their application.
KYROBIO addresses the enzyme platform challenges that have been identified as exemplar projects that contain the most significant problems facing the white biotechnology in implementing of the next generation of biocatalysts for chiral synthesis, whilst allowing the SME consortium partners to expand their marketable products and increase their technology base. These targets have been identified by matching the product needs of industry with the next generation of biocatalysts that are required to deliver these products. Industrial partners involved in KYROBIO were consulted and further discussions with the wider chemical production industry as members of CoEBio3 at UNIMAN were undertaken in the planning of KYROBIO. Consultations with industrial stakeholders have also revealed that although many new and “better” enzymes that are frequently reported in the academic literature, the fermentation science and engineering solutions coupled with economic analysis needed to implement the full supply chain for these biocatalysts is often missing and difficult to deliver. An outcome of these discussions with industrialists was that the ideal project for industry should be high input project based at a relatively limited number of academic institutes (preferably at Centres of Excellence [CoEs] that have industrial focus and relationships) with not too long a time horizon. Hence the project has a limited number of bench academic research partners, supported by 10 SMEs who are actively involved in the supradisciplinary effort required to translate research to technology development and to increase their opportunities for effective commercial exploitation of foreground knowledge. The 4 year project format with an intense level of activity is designed to meet these industrially led objectives.
It is recognized that dissemination of these results to the scientific, industrial and wider community is important to maximizing the impacts and reflecting this high priority the task of dissemination is given its own work package. This will lead to the maximum value of the project obtained by the EU funding from understanding and implementing actions needed to exploit the results beyond the lifetime of the project. It is recognised that effective engagement with wider society will be a future enabler for public acceptability and thus ultimate commercial success of industrial biotechnology. It is therefore an essential part of any biocatalysis research programme to communicate the concepts, opportunities and benefits which can flow from the application of industrial biotechnology for manufacturing. KYROBIO is expected to impact significantly on the development of a European KBBE (Knowledge Based Bioeconomy) by increasing the use of enzymes as biocatalysts. In addition KYROBIO’s technical, commercial and training/educational outputs contribute to the development of Industrial Biotechnology as a Key Enabling Technology (KET) which, going forward, provides market opportunities for sustainable use of resources, wealth creation, improved lifestyle, and employment opportunities for citizens of the EU.
KYROBIO’s broad ranging collaboration brought together expertise in biocatalyst discovery, biocatalyst design and development/ production, and process development strategies from the collective input of the participant Universities, Research Organisations and Industrial Participants.
6 major European Universities and Research centres (Universitaet Stuttgart, Danmarks Tekniske Universitet, Universitaet Graz, University of Manchester , Rijksuniversiteit Groningen, and ACIB ) and a major international chemical sector company (X-ZYM – Johnson Matthey), provide depth of knowledge in fundamentals aspects of biocatalysis and chemical manufacturing requirements. The important role of industrial participation, particularly the 10 SME participants in KYROBIO, was central to developing the novel enzymes’ further implementation into biocatalysis based technologies and products for presentation to Europe’s high-value-chemical manufacturing markets.
KYROBIO was a large and complex collaborative project, and therefore incorporated a fully integrated project structure with the full participation of enzyme- and process engineering capabilities form the project outset in order to ensure that the process requirements of end-users could be highlighted and addressed from the earliest stage of the project. This integration allowed the efficient translation of research expertise into scalable proof-of-concept experiments. The overall research methodology was subdivided into different components and followed a progression from detailed laboratory studies through process development and scale up evaluation. This research approach is designed to enable the project technical objectives to be achieved while minimising the level of risk. The use of multiple technologies with several industrial (SME) partners is seen as a key strategy to not just to spread risk and maximise impacts of the EU investment, but also to spread the exploitable benefits across the whole supply chain for development and implementation of bio catalysed processes.
The project was split into 6 research-themed Work Packages (WPs) in order to provide the necessary focus to address the challenges. Each Work Package had specialised objectives to be achieved by application of appropriate technical disciplines in defined tasks. A subset of the project participants was engaged in each WP to undertake these specialised tasks, and the WP was led by a beneficiary with extensive experience in that specific research theme. 4 out of the 6 technical Work Packages were led by SMEs - a reflection of their important leadership roles in the delivery of the project. WPs 1-4 focused on the key biocatalyst technology fields; WPs 5-6 related to the associated enabling technologies of enzyme formulation and process engineering.
Work Package 1. Enzymes for Chiral Amine Synthesis by C-N Bond Formation The principle objective of Work Package 1 is to develop C-N bond forming enzymes that can be applied for the synthesis of single enantiomer chiral amines. The chosen enzymes include transaminases, amine lyases, Pictet Spengerlases, and amine reductases all with commercial exploitation potential identified as part of the planned exploitation of the project outputs. The Work Package outputs also include the generation of libraries of enzymes presented for commercial evaluation and the synthesis at multigram scale of chiral chemicals using enzymes developed in the project.
Work Package 2.: Next Generation C-C Lyases The objectives are to deliver to the market place products from - and a portfolio of enzymes that can be applied to the synthesis of a range of single enantiomer chiral chemicals based on C-C lyases. Where this is an issue of concern, access to a Freedom to Operate (FTO) position was to be identified. This work package’s objectives are aligned to the development of Molecular Biology, enzymology, and bioinformatics tools to improve enzymes’ application to synthesis. The target enzymes are: halohydrin dehalogenase, aminotransferases, and nitrile hydratases, cyclases, and Pictet-Spenglerases.
Work Package 3 Use of molecular modelling techniques to aid the prediction of stereo chemical
reactions. A major objective here was to use molecular modelling techniques to aid the prediction of stereo chemical reactions. The data generated was used to improve reaction of substrates and lead enzyme modification programs to increase selectivity. The target enzymes included squalene cyclases and “Pictet-Spengler reaction” catalysing enzymes, hydratases and hydroxynitrilases Bioinformatics was used to identify novel enzymes for cloning and production, and novel expression and secretion methodologies were developed to aid enzyme production. .
Work Package 4. . Fermentation Science for Novel Enzymes and Improved Fermentation Strains This work package is concerned with the cloning and expression of new enzymes in suitable hosts, and in the development of production tools and systems to allow the production of KYROBIO enzymes in a timely and cost effective manner, Robust and cost effective expression and fermentation systems were developed and applied for the production of KYROBIO enzymes. Novel scalable production systems included bacterial and Pichia based biocatalyst expression systems.
Work Package 5. Enzyme and Biocatalyst Formulation for Application. The principle objective was to develop supported enzymes and enzyme formulations with improved properties (stability, activity, applicability,(recyclability) for applications and markets identified by KYROBIO. The work undertaken included stable formulation of KYROBIO enzymes, immobilised forms prepared by linking to polymer bead supports and by direct enzyme cross linking; and formulation to enable multi- enzyme processes.
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Work Package 6 Bioprocess and Chemical Engineering. This WP delivered process engineering to implement new biocatalytic processes, by tests pf selected, relatively mature enzyme systems produced in WP1-4. Efficiencies of mixing; mass transport; process control (including in situ product removal, ISPR); biointensification and biocatalyst re use were amongst the factors studied in choice of reaction design and reactor implementation. Economic evaluations of the processes and Roadmapping exercises provided recommendations for development activities for specified KYROBIO enzymes, and for broader integration of research and development activities in this field to promote efficient transition of biocatalysis from the research bench to the manufacturing facility.
Work Package 7 and Work Package 8 did not directly involve research action but focused on the supporting activities of Dissemination, Training and Exploitation Management (WP 7) and overall Project Management (WP8). The aim of KYROBIO is not only to increase the uptake of the technical project outputs with immediate stakeholders viz., the partner organisations and their clients/collaborators. A legacy of the KYROBIO project will include increased technical skills, new networks of knowledge, and increased appreciation of the interdisciplinary and inter-dependant nature of biocatalysis R&D for the consortium. The project also will inform national and international policy makers on future demands, challenges and opportunities in biotechnology research; the value of SMEs in enabling technology transfer and development; and to increase public understanding of industrial biotechnology and sustainable manufacturing issues.
Project Results:
KYROBIO has delivered new technologies for the synthesis of chiral molecules which are amongst the most important chemical building blocks for the manufacture agrochemicals, fine-chemicals and pharmaceuticals.
This has been achieved through the delivery of new enzymes and enzyme catalysed process; new tools for enzyme discovery, modification, production; and application of new process development tools and process analyses to enable their implementation. The overall research methodology was subdivided into different components and followed a progression from detailed laboratory studies through scale-up to demonstration.
The high quality research output generated in KYROBIO by the top ranking research groups operating within Europe’s leading research organisations, companies and universities, produced original research in discovery and development of new enzymes. This is reflected by the original KYROBIO research output published in 28 articles in peer reviewed journals. New and modified enzymes, cascades of enzymes and expansion of enzyme substrate range and improved selectivities was a feature of the successful outputs in the project reported by many participants notably UNIGRAZ, UNIMAN, RUG ACIB USTUTT PROZO ING and XZM. Structural analyses and bioinformatics techniques provided by RUG BIB and BPT contributed to enzyme discovery expression and modification within KYROBIO.
A notable feature of KYROBIO has been the planned access to scalability of enzyme production within the consortium and project lifetime to allow initial reaction engineering experiments, enzyme formulation and in some cases, production of products ready for commercial presentation. The project’s mix of specialist research providers in the field of biocatalyst expression and formulation (including EVO ING VTU ACIB PROZO CLEA BiCT) has also developed new methodologies and tools for biocatalyst production and formulation. These methods are not just applicable to KYROBIO enzymes, but provide a suite of new and improved technologies for exploitation by these participants well beyond the lifespan and scope of KYROBIO. This was supplemented in the project by the planned inclusion of early process engineering to integrate enzyme discovery, production, and formulation with the evolution of scalable processes, capable of ultimate commercial operation. C-Tech provided access to novel reactors for mixing and mass transfer dependent reactions, and use of electrochemical techniques to explore the impact of in situ product removal and downstream recovery of products. The availability of process modelling and analysis to identify the important metrics for future targeted improvements was an important research service provided in KYROBIO by DTU to build on the project’s technical successes and to direct future research efforts towards successful commercial scale operations.
Commercially relevant issues such as Freedom to Operate (FTO) issues were also addressed in KYROBIO. Several participants made steps to open up areas of commercially relevant research by identifying new materials and techniques to overcome some of these barriers. A case in point is the improved access to transformations based on nitrilase enzymes. At the commencement of KYROBIO there were limitations on the availability of these commercial enzyme and IP issues which made them relatively inaccessible for commercial research In collaborations between PROZO and CLEA, nitrilases identified by a metagenomics approach were also formulated by CLEA. In addition a unique nitrile hydratase enzyme obtained by fern transcriptome analysis by ACIB was also formulated as immobilised enzyme by CLEA. The project’s industrial participants have generated improved services, potential products, and openings to new markets by their involvement in the KYROBIO project. Access to wide-ranging collaboration across basic research through industrial applications has added to the breadth and depth of knowledge and networks available to all those involved in KYROBIO. A supradisciplinary approach was applied achieve these objectives combining skills in chemistry, bioinformatics, microbiology, molecular biology, structural analysis, process engineering and commercial product development amongst others. The variety of the project results and foreground outputs reflects the diversity of the research efforts, skills and organisations represented in KYROBIO. Overall, KYROBIO has delivered new knowledge and technologies with the potential to transform manufacturing processes and shape future industrial biotechnology research in Europe.
New enzymes, new chiral products
The discovery and development of new enzymes capable of generating chiral molecules through the formation of c-n and C-C bond was extensively Investigated in Work Packages 1 (C-N bond forming enzymes, led by ING), and Work Package 2 - C-C bond forming enzymes led by USTUTT. In both Work Packages academic researcher worked alongside and in frequent collaboration with SME and industrial participants to discover and develop enzymes for chiral syntheses.
Prominent amongst the activities were the development of tools for the generation of chiral amines , important building blocks for the syntheses of high value and pharmaceutical products; and the use of enzyme cascades for generation of complex molecules with specific chiral centres. Some participants explored alternative and complementary methods to generate chiral products as in the case of XZM who created various mutant libraries of both amine dehydrogenases and (S)-amine transaminases to investigate these transformations.
Application of bacterial Ammonia Lyase (EncP) and its variants to produce unnatural amino acids. (UNIMAN)
During KYROBIO researchers in UNIMAN (Nicholas Weise and Prof. Nicholas Turner) have expanded the suite of ammonia lyase enzymes as tools for the synthesis of unnatural amino acids as valuable pharmaceutical and fine chemical precursors. Investigations started with a bifunctional ammonia lyase / aminomutase, EncP from Streptomyces maritimus. The enzyme was known to take part in the biosynthesis of antibiotics but had not been developed for use in biocatalysis applications. In initial tests of the native enzyme was shown to aminate acrylic acids at the alpha and beta position. The target reaction in KYROBIO was to generate an enzyme which could perform the (S)-selective amination of arylacrylic acids at the beta-position. Using structure-guided rational redesign methods the active site of the enzyme was probed for improvement in reaction selectivity. By characterising the electronic and substrate-positioning effects of the substrates and enzyme, three variants were created with regioselectivity favouring either product. Active site engineered to reposition substrate. Each variant was characterised with respect to substrate scope and enantioselectivity and found to catalyse (S)-selective addition of ammonia across a range of 22 acrylic acid substrates. A successful the β-selective variant (EncP-R299K) was shown to catalyse the reaction desired constituting a novel biocatalyst with broad substrate scope and high enantioselectivity. The significance of the reaction is enhanced by the fact that its development completes a toolkit of enzymes derived from native EncP which can provide selective amination of arylacrylic acids at alpha or beta position with either R or S selectivity.
A portfolio of C-N bond forming enzymes for commercial applications in biocatalysis and synthetic biology ( ING)
The transaminase and ammonia lyase enzymes developed here will be implemented into ING’s programs for synthesis of API precursors and where respective amine/keto or amine/acrylate transitional functional chemical conversions are present in synthetic pathway intermediates. Furthermore, use of the transaminase and lyase variant enzymes in high throughput “life/death” screens based on assimilable nitrogen release gives rise to host growth on nitrogen free media in instances where selection tolls are required for enzyme library generation. KYROBIO also facilitated the production, formulation and immobilisation of ING’s C-N forming enzymes for process analyses development and enzyme reuse. In this project ING also developed high yielding cell density fermentation protocols to generate large quantities of transaminases and ammonia lyases cost effectively, and achieved synthetic preparation of a chiral amino acid at >100g scale using a select ammonia lyase from an achiral acid precursor
ING also took part in trials of transaminase enzymes (in conjunction with C-Tech and BiCT) to evaluate processes for intensification and product recovery by application of electrodialysis and immobilisation techniques. Unfortunately there was insufficient time remaining in the project end to complete the electrodialysis trials. However the selected process had some features which suggested improved downstream product recovery could be achieved by the electrodialysis technique, while immobilisation could remove the competing biocatalyst derived ions and biofouling issues which could reduce the efficiency of electrodialysis.
Development of a diverse range of commercial native and derivative nitrile hydratases (NHases) (PROZO)–
Unlike many other classes of biocatalysis enzymes, nitrile hydratases (NHases) are notoriously difficult to clone genes for, produce via fermentation, and stabilise for commercial application. In the KYROBIO project PROZO concentrated firstly on solving these issues, with the assistance of CLEA who have the capability to produce stable enzyme formulation for market evaluation. The labile NHase enzymes produced need to be immediately immobilised before activity is lost. The outcome of this collaboration was a rapid fundamental understanding of which NHases could be immobilised and which could not and to the production of a market leading range of 18 novel immobilised NHases with proven stability and recycling potential under typical industrial process conditions. PROZO employed its High Throughput (HT-cloning technologies were used in the challenges involved with discovering novel NHases, the company also made advancements the remaining critical area of producing highly-active NHases by bacterial fermentation. There resulted a general protocol at PROZO was developed that now enables all NHases to be activated reliably. Additionally, while studying activation of individual enzymes, a fortuitous discovery was made of a very active NHase. Unlike the other 17 NHases, this enzyme spontaneously activates, and is stable at ambient temperatures for many days in native form. PROZO’s NHase screening panel could also be interrogated by other users (i.e. customers) for specific requirements. This exposure enabled by the NHases panel, led to enzyme discovery service contracts with numerous new customers; a boost for PROZO’s growth which would not have occurred without involvement in KYROBIO.
Engineered amine dehydrogenases (AmDHs) available as part of screening kit. (XZM – JM)
XZM explored the potential of another enzyme type for the formation of C-N bonds - Amine dehydrogenases. Amine dehydrogenases can be generated from amino acid dehydrogenases by mutating 2 or more residues (leucine dehydrogenases) in the enzyme’s active site, as described in the pioneering work by Andreas Bommarius.
XZYM analysed biochemical fundamentals of this mutagenesis concept by comparing different starting point enzymes and their suitability as amine dehydrogenases. Also mutant libraries for improved kinetic parameters were generated for assessment.
The scalability of the amine dehydrogenase reaction was also studied XZYM (in collaboration with DTU), as shown and showed the aspects of reaction optimisation led to improved productivity. Choice of solvent phases and selection of stable co factor recycling schemes are factors in improving the robustness of the process. These improvements led to 94% conversion and >99% ee of the product with 100 mM substrate loading. The conversion rates were improved .3 fold y the introduction of these process improvements
Novel Transaminase variants with new substrate range available as kit and for further development (XZM - JM)
In KYROBIO, XZM, (Johnson Matthey JM) created mutants libraries based on one of its existing transaminases . One of the libraries was designed for expanding the substrate scope of one of JMs commercial kit transaminases. The wild type transaminases do not catalyse reactions of bulky-bulky amines. A small focused library of <30 transaminase variants, each containing 2- 6 mutations close to the active site was generated . Benzophenone was used as a hypothetical substrate for bioinformatic studies and to identify residues targeted in mutagenesis programme. Mutants with enhanced stereoselectivity were identified form the library (ee of the remaining R-enantiomer up to >99%). Some variants had an excessively active site, which led to >50% conversion of the resolution reaction but reduced ee of the remaining enantiomer. This case study showed the power of JMs in-house library design method for the S-transaminase enzyme family. The generated variants are also now available for screening in JM’s customer projects and form part of their commercial test kit offerings.
Decoupling of polycyclization chemistry from Alicyclobacillus acidocaldarius SHC to yield a platform for enzymatic Brønsted acid catalysis in water. (USTUTT)
USTUTT has characterized and mutated the squalene hopene cyclases from Alicyclobacillus acidocaldarius. Variants of AacSHC can perform bimolecular reactions with farnesol and small alcohols as nucleophiles revealing the potential of these enzymes for chiral Brønsted acid catalysis. Further expansion of the catalytic scope of cyclase variants by acidic activation of the pinene enabled the synthesis of various valuable monoterpenoid products. USTUTT also investigated the influence of detergents on cyclase activity (the enzymes are membrane associated).
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Enzyme cascade combining ω-Transaminases and strictosidine synthases (STR) to yield high added-value alkaloid derivatives with additional chiral centre. (UNIGRAZ)
The condensation reaction of Pictet Spengler (enzyme strictosidine synthase STR) forms the basis of biosynthesis of all monoterpenoid indole alkaloids. Possible therapeutic commercial products derived by use of the enzyme include Noscapine which is an antitussive agent and has been shown to have anticancer activity whilst (S)-Norcoclaurine is a beta-adrenergic agonist. Directed synthesis of chiral amines is of particular interest in the manufacture of drugs and other chemicals with biological activity as around 40% of all optically active drugs are chiral amines.
As part of the KYROBIO project, researchers in the team of W Kroutil (UNGRAZ) tested plant- and bacterial- derived STR enzymes for their ability to synthesis a defined isomer-3α(S)-strictosidine from tryptamine and secologanin.
Combination of Ophiorrhiza pumila STR with R or S selective ω transaminase (ωTA) in a cascade yielded products with 2 chiral centres. Various ketones and amines could be accepted as substrates by the STR. C3 absolute configuration in the product is fixed via the transaminase reaction while C1 configuration is S in all cases.
Expansion of the substrate range of the ωTA during KYROBIO enhanced the versatility of this enzyme used alone and in combination with STR in cascade reactions
KYROBIO enabled various cooperations within the consortium for development of theses enzymes and the cascade reaction. EVO and ACIB assisted on the expression of Pictet-Spenglerases, C-Tech investigated the role of electrodialysis as a process control tool to improved productivity of transaminase reactions; BIB provided valuable insight by docking and structure analysis for the strictosidine catalyzed reaction. Cooperation with UNIMAN also led to two joint publications.
Expansion of activity and substrate range of Granulicella tundricola HNL(hydroxynitrile lyase with a cupin fold) to the production of chiral cyanohydrins, important intermediates in pharmaceutical manufacturing (ACIB)
Hydroxynitrile lyases catalyse the reversible cleavage of cyanohydrins to carbonyl compounds and HCN. Recently, bacterial HNLs have been discovered in Graz, which represent a completely new type: HNLs with a cupin fold. Due to various benefits of cupins (like e.g. high yield recombinant expression in Escherichia coli, high stability) researcher at ACIB considered the class of cupin HNLs provides a new source for interesting, powerful hydroxynitrile lyases.
Unfortunately, the activity and substrate scope of the cupin HNLs known at the start of KYROBIO was quite limited (GtHNL from Granulicella tundricola (GtHNL) catalysed the synthesis of (R)-mandelonitrile with a good conversion of 80% and an enantiomeric excess of 90%). The activity and enantioselectivity of GtHNL was significantly improved by ACIB during the project by site-saturation mutagenesis of active site amino acids (designed evolution) and random mutagenesis. The combination of beneficial mutations resulted in a variant with 490-fold increased specific activity for the cyanogenesis of (R)-mandelonitrile in comparison to the wild type at the same reaction conditions. More importantly, this variant is a highly competitive alternative for the synthesis of chiral cyanohydrins, such as 2-chlorobenzaldehyde cyanohydrin, (R)-2-hydroxy-4-phenylbutyronitrile and (R)-2-hydroxy-4-phenyl-3-butene nitrile, which serve as intermediates for the synthesis of pharmaceuticals.
Novel hydroxynitrile lyases cloned and expressed by reverse genetics approach from fern transcriptome (Davallia teyermanii). (R)-cyanohydrins are produced in a highly selective manner. (ACIB)
In 1995, an HNL enzyme was published that did not resemble any known HNL but had exceptionally high specific activity. The aim in KYROBIO was to identify the primary sequence of this protein and to determine if the enzyme can be used as a biocatalyst. Therefore, the transcriptomes of a fern plant Davallia teyermanii were analysed by ACIB and matched with the peptide fragments obtained after partial purification, on-gel activity assay and mass spectroscopy. Putative hits were then investigated in the wet lab, and one of the 6 hits showed HNL activity.
With the sequence now known, the new enzyme, known as DtHNL, was heterologously expressed both in E. coli and Pichia pastoris with exceptionally high expression rates. The enzyme was also purified and the crystallized. The X-Ray data allowed to identify 6 active site residues and will be the basis for rational protein design. In addition to the first identified protein sequence, 3 isoenzymes were encoded in the Davallia teyermanii transcriptome. The four isoenzymes behave similar but not identical. The characteristics of the enzymes were studied in detail (enantioselectivity, pH optimum, stability, temperature optimum, enzyme kinetics etc.). All these results were generated by analysing the HNL enzymes in cyanogenesis direction. However, the synthetically useful reaction is the formation of a cyanohydrin. This reaction was studied with isoenzyme 1 in a biphasic system, using a small number of aldehydes and a ketone as the substrate. The HNL catalysed the formation of (R)-cyanohydrins in a highly selective manner.
DtHNLs were also produced in a glycosylated form by expression from Pichia pastoris by KYROBIO partner VTU. There are indications that glycosylation stabilizes the enzymes. Furthermore, to increase enzyme stability, KYROBIO participants CLEA Technologies produced cross linked enzyme aggregates and could show that acid stability was increased. In order to identify homologous enzymes from other origins, BPT created a 3DM database, however, the four DtHNL isoenzymes seem to form a unique subgroup and it was not possible, until now, to find similar HNL enzymes. However, further improvements can be envisaged by rational protein design based on the crystal structure that is now available.
Tools for enzyme discovery and modification
New and improved approaches to enzyme discovery and modification were achieved by development and application of structural and bioinformatics methodologies, and formed an important contribution important part of KYROBIO’s aim to improve the biocatalysis repertoire available for lyase enzyme systems.
KYROBIO has produced toolboxes for preparation of modified biocatalysts by generating complementary approaches to achieve these improvements with greater selectivity and efficiency than before. Application of advances in structural studies and their integration with bioinformatics have provided new insights into enzyme structure- function relationships. Tools for targeting individual amino acids residues or groups of amino acids for desired changes in enzyme function have been developed by several partners and was one of the major themes of Work Package 3 led by RUG. These approaches can reduce the numbers (by as much as 2 orders of magnitude), and complexity of mutated enzyme libraries to be screened for new attributes, as. This has the impact of making huge savings in we lab resources, and offering better selectivity than random or semi random approaches to library syntheses. Introducing novel enzyme functions to existing enzyme scaffold upends up new chemistry opportunities as exemplified by the decoupling of cyclisation from protonation for the enzyme squalene hopene cyclase(SHC) by researchers at USTUTT to generate variable Brønsted-acid biocatalysts capable of operating in aqueous environments.
Innovative approaches to enzyme discovery and the description of novel families of enzymes in KYROBIO has helped to overcome freedom to operate (FTO) barriers to commercial exploitation of some biocatalysis and processes. Specific examples from the project include – the adoption of metagenomics approaches to discover new enzymes ( PROZO) and the exploration of plant sources for enzyme activities with FTO (ACIB).
Establishment of innovative metagenomics program for enzyme discovery (PROZO)
In the later stages of the project PROZO collaborated once more with CLEA in the production of novel panel of nitrilases. The EU-wide enforced Nagoya Protocol (from October 2014-onwards) imposes some limits on exploitation of material (including enzymes) derived from a foreign state’s biodiversity resources the even if the intended research is only fundamental in nature. There are many potentially wide-reaching consequences of this development, especially for small SMEs. Due to these uncertainties by simply mining the public genomics databases to find enzymes may leave the researcher exposed to future royalties, and any other retrospective actions that are now planned by the EU with respect to their current sector-focused review of Nagoya Protocol implementation. PROZO therefore to initiated in-house metagenomics screens for nitrilases using UK sourced biological samples. As a result PROZO have gained significant experience amongst commercial biocatalysis enzyme suppliers with respect to the provision of metagenomically-discovered enzyme panels. These provide enzymes that are not present in the public databases, with notable advantages, where the customer benefits from a stronger IP position and unique enzyme activities, while PROZO benefits from state-of-the-art products that are very easy to market, yet protected as they are difficult to reverse engineer. In-house metagenomics-based technologies at PROZO directly resulting from KYROBIO research in this area now include (i) a large (approx. 2.5Gb) private metagenomics database, enabling new services offerings to customers focused on unique metagenomics enzyme discovery; (ii) bespoke bioinformatics software and (iii) integration with a huge and inexpensive beyond state-of-the-art cloning projects which will have capacity to clone 10000 metagenome enzymes by the end of 2016.
CorNet, a novel module of the protein superfamily analysis system 3D- an advanced tool for the analysis of correlated mutation networks. (BPT)
Based on developments in KYROBIO BPT developed and released CorNet, a novel module of the protein superfamily analysis system 3DM. 3DM is a widely used platform in many different types of protein related research. Some examples of data in a 3DM system are - structures, sequences, structure based alignments, correlated mutation data, sequence function data, ligand binding data, mutation data from literature and other data sources, SNP data from genome sequencing projects, protein flexibility data, protein-protein interaction data, and many more.
CorNet is an advanced tool for the analysis of correlated mutation networks. Application in KYROBIO showed that correlated mutation networks extracted from superfamily alignments reflect functionally relations between residues in proteins. CorNet is hyper connected to mutation data automatically extracted from the literature for all the proteins that are in the alignment behind a CorNet network. Keywords, such as specificity, activity, enantioselectivity, etc., can be used to find if mutations related to the keyword are mostly found in the positions of a CorNet network. If a keyword scores high in a specific CorNet network, then the function that is shared by the residue positions in the network has been found. In such cases the positions in the CorNet network can be used as hotspots for protein engineering In addition to its application in KYROBIO to guide the generation of ‘smart libraries’ of , the 3DM technology has wider application in guiding protein engineering projects; drug design projects, and predicting effects of human variants in DNA diagnostics.
High-throughput molecular docking method to evaluate substrate posing and scoring; yields predictions of potential enzyme substrates and products. (BIB)
Based on developments in KYROBIO BIB released a pipeline for high-throughput docking studies. The main goal of our docking-pipeline was to create a fully automated pipeline to allow high-throughput docking. Molecular docking should help address two issues: posing (the process of determining whether a given conformation and orientation of a ligand fits the active site) and scoring (the measure of the fit of a ligand into the active site). To improve posing we used 11 docking tools (Autodock Vina, Discovery Studio, Dock6, eHITS, Gemdock, Glamdock, Glide, Gold, Plants, Surflex). Additionally, in Dock6 two different algorithms were used (Dock6Flex and Dock6Rigid) and three algorithms in Discovery Studio (CDocker, DS Ligandfit, DS Libdock). To reconcile the results from different docking software we developed a novel scoring system based on comparison of the RMSD value between each poses generated in docking experiment. With these values we generated a similarity matrix and based on this we choose the best pose. Using this approach the incorrectly predicted subsets of poses should not effect on the quality of the results.
Moreover, to provide a more accurate prediction of binding affinities we use the Prime tool to calculate the binding free energy between the receptor and ligand in its complex. We estimated the binding free energy and then re-ranked the docked conformations of the best poses of protein-ligand pairs obtained from our pipeline. The Pipeline is currently being used for predicting ligand poses in Near Attack Conformation, benchmark predicted homology models, predicting potential substrates for the reaction, predicting the main product of the reaction.
Enzyme stabilization by computational library design. (RUG)
Computational design of limonene epoxide hydrolase (LEH) with engineered stereoselectivity was achieved by application of in silico methods by researchers at RUG. Improving the efficiency of mutant library construction was demonstrated by application of bioinformatics and analysis of structural information. After in silico design and ranking of 2,500 initial multi-site mutants, a small library of 37 variants was constructed which harboured nine mutant epoxide hydrolases with the desired (R,R)- or (S,S)-product selectivity. The results suggest that a large part of the experimental screening that is common in directed evolution protocols can be replaced by in silico methods. This dramatically reduces the number of physical enzyme variants required in screening experiments providing new economies of time and materials in programs of targeted enzyme improvement. The protocol for redesign of catalytic selectivity by computation (CASCO) describes a method of work for efficient enzyme redesign, of interest to commercial organisations looking to acquire specific functional changes to biocatalysts.
Biocatalyst production and formulation
Reliable and substantial enzyme production platforms are key enabling factors to opening the way for further process development, and must be considered from the earliest stages of the evolution of those processes. Without those capacities, development of processes can be difficult, and their implementation at scale impossible. New technologies for improved protein production were developed and implemented for the biocatalysts used across KYROBIO, including optimised fermentation methodologies, and improved strains and simplified media requirements for enzyme expression. New strategies for production of biocatalyst via improved expression and fermentation protocols was the focus of Work Package 4 which was led by ACIB.
Enzyme formulations and immobilisation strategies was centred on the activities in WP 5 and was led by CLEA and concentrated on formulation for application. Enzyme immobilization is a key enabling technology for the development of industrial biotransformations, facilitating the recycling and reuse of the biocatalyst and reducing contamination of product with microbe host derived substances. Cross-Linked Enzyme Aggregates (CLEAs) consist of covalently cross-linked precipitated enzymes. CLEA has developed methods for rapid immobilisation /stabilisation of enzyme in KYROBIO which can be presented to commercial clients. Bead based solid supports for immobilisation of enzymes and other biocatalyst are familiar to many researchers and are widely used in industrial applications. However a multitude of support chemistries, porosities, bead sizes, porosities and other characteristics makes it difficult of the no specialist to decide what options to consider. (BiCT)
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High level expression strains for the intracellular and secreted production of novel hydroxynitrile lyases derived form a fern species.(ACIB VTU)
Based on its broad and highly elaborated technology platform VTU Technology generated high level Pichia expression strains in KYROBIO for the intracellular and secreted production of hydroxynitrile lyases from a fern species. The corresponding enzymes were produced for stabilization and characterisation by CLEA and ACIB partner within the KYROBIO project.
New Bacterial enzyme expression platforms, stable transformants; low cost production methods (EVO)
Bacillus subtilis production strain development was successfully conducted. The application of an operon strategy has the largest impact on expression efficiency. A combination of this technology with optimized promoters and signal peptides is very promising.
Expression systems for Squalene hopene cyclase variants (USTUTT)
USTUTT has developed and optimized the expression of the squalene hopene cyclases from Zymomonas mobilis and Alicyclobacillus acidocaldarius in E. coli. The two squalene hopene cyclases genes were cloned and expressed in the pET expression system downstream of the T7 lac promoter for tightly-regulated expression in E. coli BL21(DE3) with 0.2 mM IPTG as inducer. The active recombinant proteins obtained were around 0.4-0.8 U/mg after 4 h of induction time The enzyme produced will be used in various stereospecific C-C bond forming reactions and the possibility to apply mutants of AacSHC for synthetically important organic reactions including the Prins reaction, the bimolecular nucleophilic addition of terpenoids with alcohols and acidic isomerization of pinene monoterpenes demonstrate the opportunities of this enzyme as variable Brønsted-acid biocatalysts. The diversity of Brønsted acid-catalyzed reactions addressed with SHCs allows the catalysis of a plethora of chemical reactions having no natural counterparts. Therefore, numerous applications of SHCs can be expected, providing enzymatic access to various valuable non-natural reactions. Remarkably, nature uses this acidic machinery only for polyene cyclization of di- and triterpenes. SHC provides a potent example of the potential of enzymatic catalytic centres which are by far ‘underused’ in nature, and its exploration is a powerful method to develop new catalysts.
Robust and high performing immobilized hydroxynitrile lyases (HnLs) with freedom to operate (FTO). (CLEA ACIB)
A cooperation of CLEA with ACIB and TU Graz within KYROBIO addresses the commercial availability of robust and high performing immobilized hydroxynitrile lyases (HnLs) with freedom to operate (FTO).
Immobilised nitrile hydratase CLEA panel. (PROZO CLEA)
Combining the powerful GRASP cloning technology of Prozomix with the immobilization technology of CLEA Technologies has led to the commercial availability of a large variety of robust and recyclable enzyme preparations within KYROBIO. The production route goes from the genome to a recyclable cell free biocatalyst with high operational stability in a limited amount of steps; this is important consideration for producing panels of stable enzymes with otherwise labile activity. The success of the joint effort is the availability of an immobilised nitrile hydratase CLEA panel. These nitrile hydratase CLEAs are also available in the magnetic form and are ready for presentation to commercial customers..
Nitrile hydratase are interesting biocatalysts as they have the potential to enantioselectivity hydrate nitriles to the amides under mild reaction conditions. One possible application of stereoselective conversion using these enzymes is in the production of Levetiracetam, which is a medicine against epilepsy. C-Tech collaborated with CLEA in the early stages of the KYROBIO project by developing a conventional synthetic route for a substrate to test nitrile hydratases for the relevant activity in this synthesis.
High throughput methods to develop optimised supported transaminase catalysts BiCT)
In KYROBIO BiCT used high throughput methods to develop optimised supported transaminase catalysts by balancing formulations for all the different components for each application. BiCT’s goal is to provide very cost-effective and industrial scale biocatalyst and bioprocesses. The immobilised biocatalysts are tailored on a process specific basis, to include process specific requirements for stability, and the provision of auxiliary factors such as enzymes for regeneration of cofactors. BiCT collaborated with XZM and ING on the immobilisation of their respective transaminases for stability and reuse tests carried out by BiCT. BiCT also collaborated with C-Tech on the provision of immobilised transaminase enzymes for activity and stability tests in continuous reactors including tests in plug flow systems and electrodialysis rigs operated as biocatalysis reactors.
Tools for process analysis, process control and reaction engineering
Reaction and process engineering are critical considerations for effective technology transfer from academic laboratories to the development of operable processes in a commercial setting. Use of appropriate metrics to target process improvements for economic production will improve the efficiency of biocatalyst evaluation and subsequent process development. Similarly, an often overlooked aspect of Industrial Biotechnology R&D activity is the need to improve understanding of process intensification and control, and to design methods for efficient product recovery to deliver economic benefit from biocatalysis processes. These activities were the focus of Work Package 6 which was led by C-Tech.
Economic and environmental metrics tools evaluate enzyme catalysed process to identify specific improvements to the catalyst and process, and generate recommendations for time efficient evaluation of promising biocatalysts. (DTU)
DTU’s role in KYROBIO has been economic and environmental evaluation of other partner’s biocatalytic processes to guide further development as well as benchmarking against alternative process options. The main focus has been on production of chiral amines using amine dehydrogenase, in collaboration with X ZYM. Economic evaluation has mainly been performed using economic metrics, which are economic guidelines that can be applied at an early stage of process development and reflect the cost of different parts of the process. Depending on type of product and market size different threshold values for the metrics can be been applied. To perform evaluations at an early stage of process development can guide development to save resources and direct efforts. By applying economic and environmental metrics for evaluation to the amine dehydrogenase catalysed process in KYROBIO improvements to the catalyst and process required has been identified and the tools are recommended to be applied for time-efficient evaluation of promising biocatalysts.
Lab scale electrodialysis stack reactor design and build with applications of process control and in situ product recovery demonstrated for transaminase catalysed reaction operated at 50 ml scale. (C-Tech).
Electrodialysis (ED) is an established technique for product recovery and downstream processing in large scale commercial chemical processes, environmental remediation, and fermentation science. In KYROBIO, C-Tech collaborated with UNIGRAZ to investigate the potential for electrodialysis in the early stage research lab investigations of processes-in this case a transaminase reaction. Electrodialysis exploits differences in the mobility of chemical species in an electric fields, the presence of selective membrane compartments inside the ED stack means that species can be isolated/separated by on the basis of their charge. C-Tech’s major aim was to achieve product recovery (separation of the amine product from the reaction mixture), and to alleviate coproduct inhibition by in situ removal of the inhibitory acidic ions. Wolfgang Kroutil (UNIGRAZ) kindly supplied the enzymes used in the study, information on reaction, process methodologies, and analytical techniques. As biocatalyst, (whole cell E. coli transformants expressing Chromobacterium violaceum transaminase),was in limited supply, the test reactions were carried out at 50-100 ml volume, and a small ED stack was designed and built by C-Tech to accommodate these reaction volumes. In the selected test reaction methoxyacetone is aminated to methoxy propylamine using alanine as an amine donor; pyruvate is generated as an inhibitory coproduct. After electrodialysis 80% of the amine and 97% of the pyruvate had been removed from the reaction. 68% and 82% of the current passed had been used to transport the amine and pyruvate respectively, reflecting competition by ions derived from the whole cell biocatalyst. In addition, pH control without the use of buffer salts was demonstrated. This resulted in an increased amine yield(54%) as compared to a control, salt-buffered reaction(30%). The results are encouraging as ED can be used for process control, and to reduce the amounts of salts and other reagents used in reaction - buffer salts can be eliminated and products recovered as free acids or bases.
C-Tech also collaborated with BiCT and ING to investigate the use of immobilised transaminase enzymes in small volume ED stacks operated as semi-continuous-mode biocatalysis reactors
Based on the success of work on product separations by electrodialysis initiated in KYROBIO, C-Tech has entered into two additional collaborative R&D projects to develop lab scale electrodialysis systems which are user-friendly and suitable for scientists who are new to these techniques. Applying our expertise in electrochemical cell design C-Tech have subsequently built an integrated ED unit with unique features to allow selective removal of species from reactions and help lab based researchers get started with ED as a process development and manufacturing technology.
In addition to development of electrodialysis systems for research lab applications in biocatalysis, C-Tech also evaluated various preparations (host expression systems, preparation by C-Tech and PROZO (E. coli), and ACIB (Pichia pastoris)) of monoamine oxidase (MAON-D5, mutant previously generated at UNIMAN). The whole cell biocatalyst preparations were evaluated in a model transformation of 3-amino-3-axabicyclo [3.3.0] octane in reactor systems including stirred tank (batch), shake flask and plug flow reactors. The process benefited from measures to increase oxygen supply such as the addition of molecular oxygen to the plug flow systems, which also provide enhanced mixing. Although some progress in reducing reaction time to completion was demonstrated, especially in the plug flow reactors, it proved difficult to intensify the reactions and catalyst stability remained an issue to be addressed in future work
Potential Impact:
The Potential Impact of KYROBIO
1. Socio-economic impact
The main objective of KYROBIO is to enhance the competitiveness of the European chemical industry by substituting complex chemical syntheses using multi-step (often with precious metal catalysed reactions in hazardous chemical plants) by clean and non-polluting bioprocesses and to develop advanced engineering solutions and routes to market to enable application. Meeting this objective will lead to meeting the overarching objective of enhancing the competitiveness and sustainability of the European chemical industry by replacing complex organic synthesis by cleaner biotransformation. The development and uptake of biocatalytic processes by the chemical manufacturing industry is vital for expanding the European knowledge-based bio-economy (KBBE). KYROBIO is expected to impact significantly on the development of industrial biotechnology’s contribution to the European KBBE by increasing the use of enzymes as biocatalysts for high value chemical synthesis. In addition KYROBIO’s technical, commercial and training/educational outputs contribute to the development of Industrial Biotechnology(IB) as a Key Enabling Technology (KET) which, going forward is forecast to provide market opportunities for sustainable use of resources, wealth creation, improved lifestyle, and employment opportunities for citizens of the EU.
The development of next generation biocatalysts will allow the rapid adoption of new technologies which deliver improved performance over conventional processes, with concomitant benefits to efficiency and economy. The use of enzymatic manufacturing methodologies has the potential to ‘green’ industrial chemical production by displacing conventional chemistries which require toxic and hazardous reagents, such as metal catalysts and strong acids, and energy-intensive processes operated at high temperatures and pressures. In addition to improved economies of manufacturing, this has the potential to improve the working and living condition of those involved with, or living in proximity to chemical and process industry operations. KYROBIO helped to develop the R&D skills base of the participating scientists and technologists, increase the expertise of the partner organisations, grow an active network of collaborators, and deliver new technologies for industrial application.
The significant role of industrial participant in the project, especially the SMEs was important to setting and maintaining the focus of KYROBIO in delivering its objectives. Alongside the development of novel biocatalysts, the industrial participants of KYROBIO also played a leading role in the development and applications vital enabling technologies to allow rapid development of enzymes and biocatalysts, their integration into processes and products with commercial potential, and economic and environmental evaluation versus conventional and competing technologies.
2. Societal implications
Impact on European Research Infrastructure One of successes of KYROBIO project was to gather industrial interests and research organisations from across Europe, bringing together the necessary interdisciplinary skills in order to form a strong transnational collaboration to generate new knowledge and new technologies that will be implemented in new products and technologies. KYROBIO contributed to sustain the European research and boost collaboration of industry with universities, thus allowing both European research organizations and European industries to achieve and provide innovation and leadership in these fields. The success of the project can be judged by the impressive body of high-impact literature generated, the exploitable technical foreground knowledge generated by participants (from industry and academe), and also by a lasting legacy of enhanced cross-sector collaboration. KYROBIO placed the requirements of industry at the heart of the project, leading to the development of advanced research skills to tackle real-world problems for commercializing biocatalytic reactions. An enhanced skill base will provide benefits to the research infrastructure, but also provide vital expertise for the implementation of biocatalysis by industry.
The success of KYROBIO can also be illustrated by examples of the enhanced research infrastructure which will outlive the lifespan of the project. The joint meetings and exchanges throughout the project of young researchers for training have helped them expand their skills and knowledge of industrial biocatalysis along with the critical enabling technologies, and thereby contributed to an increasing the knowledge base in Europe. Many early stage researchers in the project have experienced collaboration with industrial project partners; presented at major international conferences (including prestigious events such as BIOTRANS 2013, BIOTRANS 20I5 and The Gordon Conferences); and have continued their association with industrial biotechnology by finding related employment in academic research and commercial organisations. This next generation of scientists and technologists will be ambassadors for industrial biotechnology and from part of the extended legacy of the KYROBIO project.
Small and medium-sized enterprises (SMEs) are the backbone of Europe’s economy, providing the majority of all new jobs. All SMEs present from the projects inception and proposal formulation stage of the project have remained active in KYROBIO until its completion. The SME participants have recorded positive outcomes from the KYROBIO project ranging from securing and expanding employment; improving their research skills; enabling new technology and product development; and expansion of their international collaborative network. This is particularly striking for early stage SMEs such as PROZO and BPT who have grown staffing levels, developed products and services (e.g. novel enzyme panels, and platform tools to access bioinformatics knowledge for enzyme discovery/improvement) as a result of work carried out in KYROBIO. New technical and commercial opportunities have been identified by SME participants working in collaboration. For example, CLEA and PROZOMIX have worked together to provide a new product for their joint benefit - enzymes produced by PROZO, formulated as stabilised immobilised preparations by CLEA. C-Tech’s involvement, both as coordinator and by its technical role in KYROBIO, increased its exposure to players in the area of industrial biotechnology and opened new market opportunities for its engineering design and build capabilities. Commercially relevant outcomes such as these derived from KYROBIO strengthen the SME participants’ capability to continue to offer high value employment and commercial growth in the KBBE sector.
Continuing research collaborations have been strengthened and established, for instance, a number of KYROBIO partners (UNIMAN, USTUTT, DTU, CLEA, BiCT PROZO C-Tech and BPT) form part of the of the consortium of the FP7 project BIOOX(613849). Such examples of sustained cooperation can be found across the consortium, were individual participants have gone on to develop additional co-funded and commercial collaborations for further exploitation of concepts established in KYROBIO.
3. The main dissemination activities
The supradisciplinary approach taken in KYROBIO encourages researchers in industrial biotechnology to gain understanding and technical competence in the emerging techniques and tools for topics such as enzyme engineering and enzyme production. To this end several hands-on training courses, web-based informatics tools, webinars and other dissemination and training activities were organised as part of KYROBIO. The lab courses will be reprised post project.
Training courses. Courses in computation-supported enzyme engineering, now to be offered to PhD students and post docs on a biannual basis.(RUG) A KYROBIO masterclass held in Groningen in April 2015 was aimed to make students and post-docs familiar with the use of computational and bioinformatics tools for enzyme engineering. The improved accessibility of many tools brings them within reach of the wet biochemistry and organic chemistry labs. The program consisted of morning lectures by various speakers on basic principles of computational methods and recent highlights from their own research. Speakers from Industry and Academic backgrounds were affiliated to KYROBIO, or similar projects (BE-Basic, MicroB3, P4FIFTY) aimed at biocatalyst development.
Hands-on Pichia Lab course: "From Idea to Protein Production" was offered to academics and industrial researchers (ACIB). ACIB have provided training and knowledge transfer in practical methods for protein production, one of the key enabling technologies for biocatalyst development and application, helping to address the apparent skills shortage within this specialised area. The yeast Pichia is a useful research and industrial scale host for biocatalyst production, but remains unfamiliar to many researchers in this field.
Publications in peer reviewed journals (USTUTT, UNIGRAZ, RUG, UNIMAN, DTU, and ACIB); Webinar on Squalene Hopene Cyclases (USTUTT); Webinar highlighting KYROBIO technical achievements (KTN LTD). As could be anticipated by the high quality research output generated by the top ranking research group research organisations and universities, original research was published in 28 articles in peer reviewed journals. Further peer reviewed publications are in preparation by several KYROBIO participants. In addition, the research output afforded many opportunities for project dissemination to the wider research community at numerous international conferences. Two public webinars summarising research achievements were delivered during the project and recordings have been made available on social media and various public websites.
Presentations to civil society, including events for schoolchildren, Museum exhibits, Science Fairs and Open Day (UNIMAN). KYROBIO researchers at the University of Manchester have been heavily involved in outreach initiatives to raise awareness and widen participation in the area if industrial biotechnology. Engagement was with schools across the north of England, at the annual national science and engineering week, British science week and Manchester Institute of Biotechnology open day events. There has also been involvement in local community engagement initiatives such as Manchester science week, faculty open days and museum exhibitions. KYROBIO has also been represented at national outreach events such as the Royal Society Summer Science Exhibition 2013 and Great British Bioscience Festival 2014. Engagement activities have ranged from hands-on practical experiments and equipment demonstrations to lab tours and research talks.
International networking event, Public website and 7 project newsletters (KTN LTD). A major dissemination event was held in Brussels on 3rd Dec 2013 in conjunction with the BIONEXGEN project (266025). At the event, attended by an international audience from academia, industry and policymakers, project results were disseminated by talks; a multimedia exhibition was provided by project participants; and a panel discussion with representatives of industry and academia promoted the benefits of collaborative research in the development of Europe’s capability in Industrial Biotechnology. A publicly accessible website was established at the project commencement with information on the project and participants, news updates on project activities and general interest articles on the themes of industrial biotechnology. News and updates on project progress were disseminated to interested parties by a regular series of newsletters circulated by email and posted on the project website.
Dissemination to the widest possible audience of scientific specialists, industrial end-users, policy makers, but importantly the European public, has been at the heart of KYROBIO. By participating in outreach activities, including open days for schools, public science fairs, and engaging with diverse media (including specialist, academic trade and general press), KYROBIO researchers have helped to increase public awareness of EU research and industrial biotechnology (IB). KYROBIO researchers has been involved in the coordination and delivery of a Massive Open Online Course (MOOC) in Industrial Biotechnology as part of the University of Manchester’s research beacons initiative. The course, which is due to be released worldwide to thousands of participants during the 2016, is being designed to introduce students from diverse backgrounds to the principles of industrial biotechnology, with a focus on enzyme technology and biocatalysis for the production of pharmaceutical compounds. Submodules of the MOOC are being delivered by both internal and external academic and industrial partners, including members of KYROBIO team.
By contributing to the public understanding of IB, this project will help to improve consumer acceptance of biocatalytic manufacturing for improved functionality, safety, and environmental credentials of IB products. The enthusiastic interaction of KYROBIO scientists with young people and educators will help to increase the uptake of STEM subjects (Science, Technology, Engineering and Mathematics) by school children and students progressing to higher education, in order to nurture future generations of researchers, engineers, biocatalyst-end users, and the wider European chemical and process industries.
4. Exploitable results
New C-C and C-N bond forming biocatalysis reactions demonstrated and developed within the KYROBIO project have extended the range of chiral molecules which can be synthesised in this manner. KYROBIO has delivered new technologies for the synthesis of industrially important molecules including chiral amines, terpenoids and chiral cyanohydrins. These molecules are amongst the most important building blocks for agrochemicals, fine-chemicals and pharmaceuticals. Chirality is a critical feature in biological activity and functionality in each case. Conventional technologies for the synthetic production of chiral molecules are often wasteful and require harsh reaction conditions. Chemical routes to chiral amines can be particularly challenging, and many molecules remain completely inaccessible. Traditional manufacturing methodology for the production of chiral chemicals often require deracemisation of products, therefore limiting yields to a maximum 50%, since the unwanted enantiomer is not required.
As part of the biotechnology toolkit these enzymes play the central role in generating new products and processes. The project met the overarching challenge in this area by control of reaction stereochemistry to add value to KYROBIO chemistry. Excitingly, in many cases these new technologies, including the use of enzyme cascades in some instances, also allow access to a wider range of substrates, to generate completely novel products and processes that were not previously realisable by biocatalysis. This has the potential to not only change routes for chemical manufacture, but also to introduce new products and new functionalities in high value chemical products.
The reengineering of enzymes to generate new activities and uncoupled enzyme activities are exemplified in several instances notably in the modification off SHC enzyme by USTUTT. and generation of Amine dehydrogenase activity XZM-JM. Amine dehydrogenases can be can be generated from amino acid dehydrogenases by mutating 2 or more residues in leucine dehydrogenases. The new enzymes can supply amination of commercially interesting ketones and represent a n expansion of enzyme toolkit as an interesting complement /alternative to the use of transaminases.
The mechanism guided engineering approach of the squalene hopene cylase AacSHC will allow to gain a greater fundamental understanding in discovering new enzyme activities in the lab. By generating enzyme catalysts of Brønsted acid reactions will facilitate the synthesis of new product molecules of high added value
Applications for the new biocatalysts can be envisaged in drug- agrochemical- and speciality chemical manufactures.
• Engineered amine dehydrogenases (AmDHs) available as part of screening kit. (XZM-JM)
• Novel Transaminase variants with new substrate range available as kit and for further development (XZM-JM)
• Application of bacterial Ammonia Lyase (EncP) and its variants to produce unnatural amino acids. (UNIMAN)
• Novel terpenoid products of industrial interest produced by application of Squalene hopene cyclases (SHC) to an extended substrate range. (USTUTT)
• Decoupling of polycyclization chemistry from Alicyclobacillus acidocaldarius SHC to yield a platform for enzymatic Brønsted acid catalysis in water. (USTUTT)
• Enzyme cascade combining ω-Transaminases and strictosidine synthases to yield high added-value alkaloid derivatives with additional chiral centre. (UNIGRAZ)
• Expansion of activity and substrate range of Granulicella tundricola HNL(hydroxynitrile lyase with a cupin fold) to the production of chiral cyanohydrins, important intermediates in pharmaceutical manufacturing (ACIB)
• Novel hydroxynitrile lyases cloned and expressed by reverse genetics approach from fern transcriptome (Davallia teyermanii). (R)-cyanohydrins are produced in a highly selective manner. (ACIB)
Tools for enzyme discovery and modification, frequently achieved by development and application of structural and bioinformatics methodologies, was an important part if KYROBIO’s aim to improve the biocatalysis (product) repertoire available for enzyme systems. KYROBIO has produced toolboxes for preparation of modified biocatalysts by generating complementary approaches to achieve these improvements with greater selectivity and efficiency than before. Innovative approaches to enzyme discovery and the description of novel families of enzymes in KYROBIO has helped to overcome freedom to operate (FTO) barriers to commercial exploitation of some biocatalysis and processes. Specific examples from the project include -
• Establishment of innovative metagenomics program for enzyme discovery (PROZO)
• CorNet, a novel module of the protein superfamily analysis system 3DM- an advanced tool for the analysis of correlated mutation networks. (BPT)
• High-throughput molecular docking method to evaluate substrate posing and scoring; yields predictions of potential enzyme substrates and products. (BIB)
• Efficient enzyme modification protocols by computational library design. (RUG)
Reliable and substantial enzyme production platforms are key enabling factors to opening the way for further process development, and must be considered from the earliest stages of the evolution of those processes. Examples of improved/exploitable microbial enzyme production systems were demonstrated in KYROBIO including productions of plant derived HNL enzyme in Pichia pastoris (VTU); methanol-free enzyme expression in Pichia pastoris (ACIB); and stable chromosomal based enzyme expression and secretion in Bacillus (EVO). Improved enzyme production systems have multiple positive economic and environmental impacts. These improvements in enzyme production could production costs by reducing materials and energy usage. Moving away from promotor systems which are dependent on inducer compounds (methanol in the case of Pichia) and selection systems which require the presence of antibiotics reduce both material cost and environmental burden of enzyme production. Specific examples of exploitable KYROBIO outputs in this area include -
• High level expression strains (Pichia) for the intracellular and secreted production of novel hydroxynitrile lyases derived form a fern species.(VTU)
• New Bacillus host and expression systems for stable enzyme expression and secretion of enzymes.
Enzyme immobilization is a key enabling technology for the development of industrial biotransformations, facilitating the recycling and reuse of the biocatalyst, as well as offering improvements in catalyst stability. Cross-linked Enzyme Aggregates (CLEAs) consist of covalently cross-linked precipitated enzymes. CLEA has developed methods for rapid immobilisation /stabilisation of enzyme in KYROBIO which can be presented to commercial clients. Bead-based solid supports for the immobilisations of enzymes are available in many formats varying in many aspects including diameter, porosity, stability and method of linkage. Tailoring the immobilisation methodology to the process application needs has been undertaken by scientists at BiCT to produce a suite of immobilised transaminase enzymes developed within KYROBIO.
• Robust and high performing immobilized hydroxynitrile lyases (HnLs) with freedom to operate (FTO). (CLEA ACIB)
• Immobilised nitrile hydratase CLEA panel. (PROZO CLEA)
• High throughput methods to develop optimised supported transaminase catalysts by balancing formulations for all the different components for each application. (BICT)
Process engineering and tools for process design are critical considerations for effective technology transfer from academic laboratories to the development of operable processes in a commercial setting. Use of appropriate metrics to target process improvements for economic production will improve the efficiency of biocatalyst evaluation and subsequent process development. Similarly, an often overlooked aspect of IB R&D activity is the need to improve understanding of process intensification and control, and to design methods for efficient product recovery to deliver economic benefit from biocatalysis processes. Specific examples of exploitable KYROBIO outputs in this area include -
• Economic and environmental metrics tools evaluate enzyme catalysed process to identify specific improvements to the catalyst and process, and generate recommendations for time efficient evaluation of promising biocatalysts. (DTU).
• Lab scale electrodialysis stack reactor design and build with applications of process control and in situ product recovery demonstrated for transaminase catalysed reaction operated at 50 ml scale. (C-Tech)
List of Websites:
www.kyrobio.eu
The 4-year KYROBIO project aimed to improve the range of high value single enantiomer chiral chemicals manufactured in Europe using biotechnological routes. The use of industrial biotechnology for the production of chiral chemicals has already shown great potential. Effective competition with traditional chemo catalysts has been achieved in many instances, through specific targeted synthesis of the enantiomer of choice, thus increasing the total production yield of product. However biocatalysis methodologies still havegaps within its capabilities, when compared to the breadth of chemo-catalysis. The KYROBIO project is filling some of these gaps by developing new classes of enzyme catalysts and the associated process and economic knowledge needed for their exploitation.
The principle focus of research effort in KYROBIO was to enable enabling the industrial application of the lyase class of enzymes. Lyase enzymes have potential to expand the application of biocatalysis in chemical synthesis - they can selectively synthesize molecules with multiple chiral centres, based on the formation of carbon-carbon and carbon-nitrogen bonds. Increasing the availability and applicability of a variety of novel lyase enzymes suitable for industrial applications was a central objective of the KYROBIO project.
The development and application of these biocatalysts was supported by many technical disciplines working in concert. Technical approaches integrated enzyme discovery strategies; bioinformatics; structural studies; enzyme production platforms (fermentation, expression); enzyme process engineering; and biocatalyst formulation. This multidisciplinary approach was developed to expand and enhance the ranges of technical tools and skills needed to establish both the biocatalyst’s credentials and the supporting technologies to open exploitation routes for application of enzymes in the manufacture of high value chemical products.
A strong emphasis on dissemination from the project outset provided engagement with both specialist audiences and the wider public. A programme of networking events, publications and online communications, webinars, public engagement and specialised training events has been delivered. A major dissemination event held in Brussels to disseminate the project results and to promote the benefits of collaborative research in the development of Europe’s’ capability in Industrial Biotechnology. This was augmented by publication of original research in prestigious journals, and presentations at international conferences. Wider audiences were addressed in Science Open Days, Museum exhibitions, and networking events aimed at informing and engaging school children with biotechnology-themed topics.
The KYROBIO project has a 17 -member consortium comprising 5 Universities, 11 SMEs and a large multinational company. Complementary technical and commercial skills represented in the consortium have been a major project strength and have facilitated close collaborations to allow mutual exchange of fundamental knowledge, applied research techniques and technology development processes. KYROBIO’s largely industry focussed approach, coupled with strong SME participation and leadership, has been used to address industrially-identified needs for chiral synthesis using biocatalysis, and supply the necessary supporting technologies. Exploitable outputs include - novel biotransformations of interest to speciality chemical and pharmaceutical manufacturers; new technical platforms for enzyme discovery; bioinformatics/ structural based tools for enzyme improvements; expression and fermentation techniques to enhance biocatalyst manufacturing; and process and product recovery technologies to increase efficiency of biocatalysis processes. The strong industrial focus of the project has been successful in fully engaging participants who can exploit the added value of biocatalysis. At the project end date (30 Nov 2015) participants have taken some of the output from the KYROBIO project to market, and have advanced plans for further exploitation of the knowledge and materials developed within the project to generate new competitive products and services for the European chemical manufacturing industry.
Beneficiaries C-TECH INNOVATION LTD (Coordinator), evocatal GmbH , BiCT SRL, Ingenza LTD,CLEA TECHNOLOGIES BV, BIOINFOBANK INSTITUTE, PROZOMIX LIMITED, VTU TECHNOLOGY GMBH, x- ZYME GMBH,BIO-PRODICT BV, ACIB GMBH, UNIVERSITAET GRAZ UNIVERSITY OF MANCHESTER, RIJKSUNIVERSITEIT GRONINGEN, DANMARKS TEKNISKE UNIVERSITET, UNIVERSITAET STUTTGART, CI-KTN (now KTN LTD).
Project Context and Objectives:
The KYROBIO project was planned and delivered with the objective to generate a toolbox of new enzymes and technologies that could be used to synthesize single enantiomer chiral chemicals to be produced by industry in Europe using biocatalytic routes.
The main objective of KYROBIO was to enhance the competitiveness of the European chemical industry by substituting complex chemical syntheses using multi-step (often with precious metal catalysed reactions in hazardous chemical plants) by clean and non-polluting bioprocesses and to develop advanced engineering solutions and routes to market to enable their application.
KYROBIO addresses the enzyme platform challenges that have been identified as exemplar projects that contain the most significant problems facing the white biotechnology in implementing of the next generation of biocatalysts for chiral synthesis, whilst allowing the SME consortium partners to expand their marketable products and increase their technology base. These targets have been identified by matching the product needs of industry with the next generation of biocatalysts that are required to deliver these products. Industrial partners involved in KYROBIO were consulted and further discussions with the wider chemical production industry as members of CoEBio3 at UNIMAN were undertaken in the planning of KYROBIO. Consultations with industrial stakeholders have also revealed that although many new and “better” enzymes that are frequently reported in the academic literature, the fermentation science and engineering solutions coupled with economic analysis needed to implement the full supply chain for these biocatalysts is often missing and difficult to deliver. An outcome of these discussions with industrialists was that the ideal project for industry should be high input project based at a relatively limited number of academic institutes (preferably at Centres of Excellence [CoEs] that have industrial focus and relationships) with not too long a time horizon. Hence the project has a limited number of bench academic research partners, supported by 10 SMEs who are actively involved in the supradisciplinary effort required to translate research to technology development and to increase their opportunities for effective commercial exploitation of foreground knowledge. The 4 year project format with an intense level of activity is designed to meet these industrially led objectives.
It is recognized that dissemination of these results to the scientific, industrial and wider community is important to maximizing the impacts and reflecting this high priority the task of dissemination is given its own work package. This will lead to the maximum value of the project obtained by the EU funding from understanding and implementing actions needed to exploit the results beyond the lifetime of the project. It is recognised that effective engagement with wider society will be a future enabler for public acceptability and thus ultimate commercial success of industrial biotechnology. It is therefore an essential part of any biocatalysis research programme to communicate the concepts, opportunities and benefits which can flow from the application of industrial biotechnology for manufacturing. KYROBIO is expected to impact significantly on the development of a European KBBE (Knowledge Based Bioeconomy) by increasing the use of enzymes as biocatalysts. In addition KYROBIO’s technical, commercial and training/educational outputs contribute to the development of Industrial Biotechnology as a Key Enabling Technology (KET) which, going forward, provides market opportunities for sustainable use of resources, wealth creation, improved lifestyle, and employment opportunities for citizens of the EU.
KYROBIO’s broad ranging collaboration brought together expertise in biocatalyst discovery, biocatalyst design and development/ production, and process development strategies from the collective input of the participant Universities, Research Organisations and Industrial Participants.
6 major European Universities and Research centres (Universitaet Stuttgart, Danmarks Tekniske Universitet, Universitaet Graz, University of Manchester , Rijksuniversiteit Groningen, and ACIB ) and a major international chemical sector company (X-ZYM – Johnson Matthey), provide depth of knowledge in fundamentals aspects of biocatalysis and chemical manufacturing requirements. The important role of industrial participation, particularly the 10 SME participants in KYROBIO, was central to developing the novel enzymes’ further implementation into biocatalysis based technologies and products for presentation to Europe’s high-value-chemical manufacturing markets.
KYROBIO was a large and complex collaborative project, and therefore incorporated a fully integrated project structure with the full participation of enzyme- and process engineering capabilities form the project outset in order to ensure that the process requirements of end-users could be highlighted and addressed from the earliest stage of the project. This integration allowed the efficient translation of research expertise into scalable proof-of-concept experiments. The overall research methodology was subdivided into different components and followed a progression from detailed laboratory studies through process development and scale up evaluation. This research approach is designed to enable the project technical objectives to be achieved while minimising the level of risk. The use of multiple technologies with several industrial (SME) partners is seen as a key strategy to not just to spread risk and maximise impacts of the EU investment, but also to spread the exploitable benefits across the whole supply chain for development and implementation of bio catalysed processes.
The project was split into 6 research-themed Work Packages (WPs) in order to provide the necessary focus to address the challenges. Each Work Package had specialised objectives to be achieved by application of appropriate technical disciplines in defined tasks. A subset of the project participants was engaged in each WP to undertake these specialised tasks, and the WP was led by a beneficiary with extensive experience in that specific research theme. 4 out of the 6 technical Work Packages were led by SMEs - a reflection of their important leadership roles in the delivery of the project. WPs 1-4 focused on the key biocatalyst technology fields; WPs 5-6 related to the associated enabling technologies of enzyme formulation and process engineering.
Work Package 1. Enzymes for Chiral Amine Synthesis by C-N Bond Formation The principle objective of Work Package 1 is to develop C-N bond forming enzymes that can be applied for the synthesis of single enantiomer chiral amines. The chosen enzymes include transaminases, amine lyases, Pictet Spengerlases, and amine reductases all with commercial exploitation potential identified as part of the planned exploitation of the project outputs. The Work Package outputs also include the generation of libraries of enzymes presented for commercial evaluation and the synthesis at multigram scale of chiral chemicals using enzymes developed in the project.
Work Package 2.: Next Generation C-C Lyases The objectives are to deliver to the market place products from - and a portfolio of enzymes that can be applied to the synthesis of a range of single enantiomer chiral chemicals based on C-C lyases. Where this is an issue of concern, access to a Freedom to Operate (FTO) position was to be identified. This work package’s objectives are aligned to the development of Molecular Biology, enzymology, and bioinformatics tools to improve enzymes’ application to synthesis. The target enzymes are: halohydrin dehalogenase, aminotransferases, and nitrile hydratases, cyclases, and Pictet-Spenglerases.
Work Package 3 Use of molecular modelling techniques to aid the prediction of stereo chemical
reactions. A major objective here was to use molecular modelling techniques to aid the prediction of stereo chemical reactions. The data generated was used to improve reaction of substrates and lead enzyme modification programs to increase selectivity. The target enzymes included squalene cyclases and “Pictet-Spengler reaction” catalysing enzymes, hydratases and hydroxynitrilases Bioinformatics was used to identify novel enzymes for cloning and production, and novel expression and secretion methodologies were developed to aid enzyme production. .
Work Package 4. . Fermentation Science for Novel Enzymes and Improved Fermentation Strains This work package is concerned with the cloning and expression of new enzymes in suitable hosts, and in the development of production tools and systems to allow the production of KYROBIO enzymes in a timely and cost effective manner, Robust and cost effective expression and fermentation systems were developed and applied for the production of KYROBIO enzymes. Novel scalable production systems included bacterial and Pichia based biocatalyst expression systems.
Work Package 5. Enzyme and Biocatalyst Formulation for Application. The principle objective was to develop supported enzymes and enzyme formulations with improved properties (stability, activity, applicability,(recyclability) for applications and markets identified by KYROBIO. The work undertaken included stable formulation of KYROBIO enzymes, immobilised forms prepared by linking to polymer bead supports and by direct enzyme cross linking; and formulation to enable multi- enzyme processes.
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Work Package 6 Bioprocess and Chemical Engineering. This WP delivered process engineering to implement new biocatalytic processes, by tests pf selected, relatively mature enzyme systems produced in WP1-4. Efficiencies of mixing; mass transport; process control (including in situ product removal, ISPR); biointensification and biocatalyst re use were amongst the factors studied in choice of reaction design and reactor implementation. Economic evaluations of the processes and Roadmapping exercises provided recommendations for development activities for specified KYROBIO enzymes, and for broader integration of research and development activities in this field to promote efficient transition of biocatalysis from the research bench to the manufacturing facility.
Work Package 7 and Work Package 8 did not directly involve research action but focused on the supporting activities of Dissemination, Training and Exploitation Management (WP 7) and overall Project Management (WP8). The aim of KYROBIO is not only to increase the uptake of the technical project outputs with immediate stakeholders viz., the partner organisations and their clients/collaborators. A legacy of the KYROBIO project will include increased technical skills, new networks of knowledge, and increased appreciation of the interdisciplinary and inter-dependant nature of biocatalysis R&D for the consortium. The project also will inform national and international policy makers on future demands, challenges and opportunities in biotechnology research; the value of SMEs in enabling technology transfer and development; and to increase public understanding of industrial biotechnology and sustainable manufacturing issues.
Project Results:
KYROBIO has delivered new technologies for the synthesis of chiral molecules which are amongst the most important chemical building blocks for the manufacture agrochemicals, fine-chemicals and pharmaceuticals.
This has been achieved through the delivery of new enzymes and enzyme catalysed process; new tools for enzyme discovery, modification, production; and application of new process development tools and process analyses to enable their implementation. The overall research methodology was subdivided into different components and followed a progression from detailed laboratory studies through scale-up to demonstration.
The high quality research output generated in KYROBIO by the top ranking research groups operating within Europe’s leading research organisations, companies and universities, produced original research in discovery and development of new enzymes. This is reflected by the original KYROBIO research output published in 28 articles in peer reviewed journals. New and modified enzymes, cascades of enzymes and expansion of enzyme substrate range and improved selectivities was a feature of the successful outputs in the project reported by many participants notably UNIGRAZ, UNIMAN, RUG ACIB USTUTT PROZO ING and XZM. Structural analyses and bioinformatics techniques provided by RUG BIB and BPT contributed to enzyme discovery expression and modification within KYROBIO.
A notable feature of KYROBIO has been the planned access to scalability of enzyme production within the consortium and project lifetime to allow initial reaction engineering experiments, enzyme formulation and in some cases, production of products ready for commercial presentation. The project’s mix of specialist research providers in the field of biocatalyst expression and formulation (including EVO ING VTU ACIB PROZO CLEA BiCT) has also developed new methodologies and tools for biocatalyst production and formulation. These methods are not just applicable to KYROBIO enzymes, but provide a suite of new and improved technologies for exploitation by these participants well beyond the lifespan and scope of KYROBIO. This was supplemented in the project by the planned inclusion of early process engineering to integrate enzyme discovery, production, and formulation with the evolution of scalable processes, capable of ultimate commercial operation. C-Tech provided access to novel reactors for mixing and mass transfer dependent reactions, and use of electrochemical techniques to explore the impact of in situ product removal and downstream recovery of products. The availability of process modelling and analysis to identify the important metrics for future targeted improvements was an important research service provided in KYROBIO by DTU to build on the project’s technical successes and to direct future research efforts towards successful commercial scale operations.
Commercially relevant issues such as Freedom to Operate (FTO) issues were also addressed in KYROBIO. Several participants made steps to open up areas of commercially relevant research by identifying new materials and techniques to overcome some of these barriers. A case in point is the improved access to transformations based on nitrilase enzymes. At the commencement of KYROBIO there were limitations on the availability of these commercial enzyme and IP issues which made them relatively inaccessible for commercial research In collaborations between PROZO and CLEA, nitrilases identified by a metagenomics approach were also formulated by CLEA. In addition a unique nitrile hydratase enzyme obtained by fern transcriptome analysis by ACIB was also formulated as immobilised enzyme by CLEA. The project’s industrial participants have generated improved services, potential products, and openings to new markets by their involvement in the KYROBIO project. Access to wide-ranging collaboration across basic research through industrial applications has added to the breadth and depth of knowledge and networks available to all those involved in KYROBIO. A supradisciplinary approach was applied achieve these objectives combining skills in chemistry, bioinformatics, microbiology, molecular biology, structural analysis, process engineering and commercial product development amongst others. The variety of the project results and foreground outputs reflects the diversity of the research efforts, skills and organisations represented in KYROBIO. Overall, KYROBIO has delivered new knowledge and technologies with the potential to transform manufacturing processes and shape future industrial biotechnology research in Europe.
New enzymes, new chiral products
The discovery and development of new enzymes capable of generating chiral molecules through the formation of c-n and C-C bond was extensively Investigated in Work Packages 1 (C-N bond forming enzymes, led by ING), and Work Package 2 - C-C bond forming enzymes led by USTUTT. In both Work Packages academic researcher worked alongside and in frequent collaboration with SME and industrial participants to discover and develop enzymes for chiral syntheses.
Prominent amongst the activities were the development of tools for the generation of chiral amines , important building blocks for the syntheses of high value and pharmaceutical products; and the use of enzyme cascades for generation of complex molecules with specific chiral centres. Some participants explored alternative and complementary methods to generate chiral products as in the case of XZM who created various mutant libraries of both amine dehydrogenases and (S)-amine transaminases to investigate these transformations.
Application of bacterial Ammonia Lyase (EncP) and its variants to produce unnatural amino acids. (UNIMAN)
During KYROBIO researchers in UNIMAN (Nicholas Weise and Prof. Nicholas Turner) have expanded the suite of ammonia lyase enzymes as tools for the synthesis of unnatural amino acids as valuable pharmaceutical and fine chemical precursors. Investigations started with a bifunctional ammonia lyase / aminomutase, EncP from Streptomyces maritimus. The enzyme was known to take part in the biosynthesis of antibiotics but had not been developed for use in biocatalysis applications. In initial tests of the native enzyme was shown to aminate acrylic acids at the alpha and beta position. The target reaction in KYROBIO was to generate an enzyme which could perform the (S)-selective amination of arylacrylic acids at the beta-position. Using structure-guided rational redesign methods the active site of the enzyme was probed for improvement in reaction selectivity. By characterising the electronic and substrate-positioning effects of the substrates and enzyme, three variants were created with regioselectivity favouring either product. Active site engineered to reposition substrate. Each variant was characterised with respect to substrate scope and enantioselectivity and found to catalyse (S)-selective addition of ammonia across a range of 22 acrylic acid substrates. A successful the β-selective variant (EncP-R299K) was shown to catalyse the reaction desired constituting a novel biocatalyst with broad substrate scope and high enantioselectivity. The significance of the reaction is enhanced by the fact that its development completes a toolkit of enzymes derived from native EncP which can provide selective amination of arylacrylic acids at alpha or beta position with either R or S selectivity.
A portfolio of C-N bond forming enzymes for commercial applications in biocatalysis and synthetic biology ( ING)
The transaminase and ammonia lyase enzymes developed here will be implemented into ING’s programs for synthesis of API precursors and where respective amine/keto or amine/acrylate transitional functional chemical conversions are present in synthetic pathway intermediates. Furthermore, use of the transaminase and lyase variant enzymes in high throughput “life/death” screens based on assimilable nitrogen release gives rise to host growth on nitrogen free media in instances where selection tolls are required for enzyme library generation. KYROBIO also facilitated the production, formulation and immobilisation of ING’s C-N forming enzymes for process analyses development and enzyme reuse. In this project ING also developed high yielding cell density fermentation protocols to generate large quantities of transaminases and ammonia lyases cost effectively, and achieved synthetic preparation of a chiral amino acid at >100g scale using a select ammonia lyase from an achiral acid precursor
ING also took part in trials of transaminase enzymes (in conjunction with C-Tech and BiCT) to evaluate processes for intensification and product recovery by application of electrodialysis and immobilisation techniques. Unfortunately there was insufficient time remaining in the project end to complete the electrodialysis trials. However the selected process had some features which suggested improved downstream product recovery could be achieved by the electrodialysis technique, while immobilisation could remove the competing biocatalyst derived ions and biofouling issues which could reduce the efficiency of electrodialysis.
Development of a diverse range of commercial native and derivative nitrile hydratases (NHases) (PROZO)–
Unlike many other classes of biocatalysis enzymes, nitrile hydratases (NHases) are notoriously difficult to clone genes for, produce via fermentation, and stabilise for commercial application. In the KYROBIO project PROZO concentrated firstly on solving these issues, with the assistance of CLEA who have the capability to produce stable enzyme formulation for market evaluation. The labile NHase enzymes produced need to be immediately immobilised before activity is lost. The outcome of this collaboration was a rapid fundamental understanding of which NHases could be immobilised and which could not and to the production of a market leading range of 18 novel immobilised NHases with proven stability and recycling potential under typical industrial process conditions. PROZO employed its High Throughput (HT-cloning technologies were used in the challenges involved with discovering novel NHases, the company also made advancements the remaining critical area of producing highly-active NHases by bacterial fermentation. There resulted a general protocol at PROZO was developed that now enables all NHases to be activated reliably. Additionally, while studying activation of individual enzymes, a fortuitous discovery was made of a very active NHase. Unlike the other 17 NHases, this enzyme spontaneously activates, and is stable at ambient temperatures for many days in native form. PROZO’s NHase screening panel could also be interrogated by other users (i.e. customers) for specific requirements. This exposure enabled by the NHases panel, led to enzyme discovery service contracts with numerous new customers; a boost for PROZO’s growth which would not have occurred without involvement in KYROBIO.
Engineered amine dehydrogenases (AmDHs) available as part of screening kit. (XZM – JM)
XZM explored the potential of another enzyme type for the formation of C-N bonds - Amine dehydrogenases. Amine dehydrogenases can be generated from amino acid dehydrogenases by mutating 2 or more residues (leucine dehydrogenases) in the enzyme’s active site, as described in the pioneering work by Andreas Bommarius.
XZYM analysed biochemical fundamentals of this mutagenesis concept by comparing different starting point enzymes and their suitability as amine dehydrogenases. Also mutant libraries for improved kinetic parameters were generated for assessment.
The scalability of the amine dehydrogenase reaction was also studied XZYM (in collaboration with DTU), as shown and showed the aspects of reaction optimisation led to improved productivity. Choice of solvent phases and selection of stable co factor recycling schemes are factors in improving the robustness of the process. These improvements led to 94% conversion and >99% ee of the product with 100 mM substrate loading. The conversion rates were improved .3 fold y the introduction of these process improvements
Novel Transaminase variants with new substrate range available as kit and for further development (XZM - JM)
In KYROBIO, XZM, (Johnson Matthey JM) created mutants libraries based on one of its existing transaminases . One of the libraries was designed for expanding the substrate scope of one of JMs commercial kit transaminases. The wild type transaminases do not catalyse reactions of bulky-bulky amines. A small focused library of <30 transaminase variants, each containing 2- 6 mutations close to the active site was generated . Benzophenone was used as a hypothetical substrate for bioinformatic studies and to identify residues targeted in mutagenesis programme. Mutants with enhanced stereoselectivity were identified form the library (ee of the remaining R-enantiomer up to >99%). Some variants had an excessively active site, which led to >50% conversion of the resolution reaction but reduced ee of the remaining enantiomer. This case study showed the power of JMs in-house library design method for the S-transaminase enzyme family. The generated variants are also now available for screening in JM’s customer projects and form part of their commercial test kit offerings.
Decoupling of polycyclization chemistry from Alicyclobacillus acidocaldarius SHC to yield a platform for enzymatic Brønsted acid catalysis in water. (USTUTT)
USTUTT has characterized and mutated the squalene hopene cyclases from Alicyclobacillus acidocaldarius. Variants of AacSHC can perform bimolecular reactions with farnesol and small alcohols as nucleophiles revealing the potential of these enzymes for chiral Brønsted acid catalysis. Further expansion of the catalytic scope of cyclase variants by acidic activation of the pinene enabled the synthesis of various valuable monoterpenoid products. USTUTT also investigated the influence of detergents on cyclase activity (the enzymes are membrane associated).
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Enzyme cascade combining ω-Transaminases and strictosidine synthases (STR) to yield high added-value alkaloid derivatives with additional chiral centre. (UNIGRAZ)
The condensation reaction of Pictet Spengler (enzyme strictosidine synthase STR) forms the basis of biosynthesis of all monoterpenoid indole alkaloids. Possible therapeutic commercial products derived by use of the enzyme include Noscapine which is an antitussive agent and has been shown to have anticancer activity whilst (S)-Norcoclaurine is a beta-adrenergic agonist. Directed synthesis of chiral amines is of particular interest in the manufacture of drugs and other chemicals with biological activity as around 40% of all optically active drugs are chiral amines.
As part of the KYROBIO project, researchers in the team of W Kroutil (UNGRAZ) tested plant- and bacterial- derived STR enzymes for their ability to synthesis a defined isomer-3α(S)-strictosidine from tryptamine and secologanin.
Combination of Ophiorrhiza pumila STR with R or S selective ω transaminase (ωTA) in a cascade yielded products with 2 chiral centres. Various ketones and amines could be accepted as substrates by the STR. C3 absolute configuration in the product is fixed via the transaminase reaction while C1 configuration is S in all cases.
Expansion of the substrate range of the ωTA during KYROBIO enhanced the versatility of this enzyme used alone and in combination with STR in cascade reactions
KYROBIO enabled various cooperations within the consortium for development of theses enzymes and the cascade reaction. EVO and ACIB assisted on the expression of Pictet-Spenglerases, C-Tech investigated the role of electrodialysis as a process control tool to improved productivity of transaminase reactions; BIB provided valuable insight by docking and structure analysis for the strictosidine catalyzed reaction. Cooperation with UNIMAN also led to two joint publications.
Expansion of activity and substrate range of Granulicella tundricola HNL(hydroxynitrile lyase with a cupin fold) to the production of chiral cyanohydrins, important intermediates in pharmaceutical manufacturing (ACIB)
Hydroxynitrile lyases catalyse the reversible cleavage of cyanohydrins to carbonyl compounds and HCN. Recently, bacterial HNLs have been discovered in Graz, which represent a completely new type: HNLs with a cupin fold. Due to various benefits of cupins (like e.g. high yield recombinant expression in Escherichia coli, high stability) researcher at ACIB considered the class of cupin HNLs provides a new source for interesting, powerful hydroxynitrile lyases.
Unfortunately, the activity and substrate scope of the cupin HNLs known at the start of KYROBIO was quite limited (GtHNL from Granulicella tundricola (GtHNL) catalysed the synthesis of (R)-mandelonitrile with a good conversion of 80% and an enantiomeric excess of 90%). The activity and enantioselectivity of GtHNL was significantly improved by ACIB during the project by site-saturation mutagenesis of active site amino acids (designed evolution) and random mutagenesis. The combination of beneficial mutations resulted in a variant with 490-fold increased specific activity for the cyanogenesis of (R)-mandelonitrile in comparison to the wild type at the same reaction conditions. More importantly, this variant is a highly competitive alternative for the synthesis of chiral cyanohydrins, such as 2-chlorobenzaldehyde cyanohydrin, (R)-2-hydroxy-4-phenylbutyronitrile and (R)-2-hydroxy-4-phenyl-3-butene nitrile, which serve as intermediates for the synthesis of pharmaceuticals.
Novel hydroxynitrile lyases cloned and expressed by reverse genetics approach from fern transcriptome (Davallia teyermanii). (R)-cyanohydrins are produced in a highly selective manner. (ACIB)
In 1995, an HNL enzyme was published that did not resemble any known HNL but had exceptionally high specific activity. The aim in KYROBIO was to identify the primary sequence of this protein and to determine if the enzyme can be used as a biocatalyst. Therefore, the transcriptomes of a fern plant Davallia teyermanii were analysed by ACIB and matched with the peptide fragments obtained after partial purification, on-gel activity assay and mass spectroscopy. Putative hits were then investigated in the wet lab, and one of the 6 hits showed HNL activity.
With the sequence now known, the new enzyme, known as DtHNL, was heterologously expressed both in E. coli and Pichia pastoris with exceptionally high expression rates. The enzyme was also purified and the crystallized. The X-Ray data allowed to identify 6 active site residues and will be the basis for rational protein design. In addition to the first identified protein sequence, 3 isoenzymes were encoded in the Davallia teyermanii transcriptome. The four isoenzymes behave similar but not identical. The characteristics of the enzymes were studied in detail (enantioselectivity, pH optimum, stability, temperature optimum, enzyme kinetics etc.). All these results were generated by analysing the HNL enzymes in cyanogenesis direction. However, the synthetically useful reaction is the formation of a cyanohydrin. This reaction was studied with isoenzyme 1 in a biphasic system, using a small number of aldehydes and a ketone as the substrate. The HNL catalysed the formation of (R)-cyanohydrins in a highly selective manner.
DtHNLs were also produced in a glycosylated form by expression from Pichia pastoris by KYROBIO partner VTU. There are indications that glycosylation stabilizes the enzymes. Furthermore, to increase enzyme stability, KYROBIO participants CLEA Technologies produced cross linked enzyme aggregates and could show that acid stability was increased. In order to identify homologous enzymes from other origins, BPT created a 3DM database, however, the four DtHNL isoenzymes seem to form a unique subgroup and it was not possible, until now, to find similar HNL enzymes. However, further improvements can be envisaged by rational protein design based on the crystal structure that is now available.
Tools for enzyme discovery and modification
New and improved approaches to enzyme discovery and modification were achieved by development and application of structural and bioinformatics methodologies, and formed an important contribution important part of KYROBIO’s aim to improve the biocatalysis repertoire available for lyase enzyme systems.
KYROBIO has produced toolboxes for preparation of modified biocatalysts by generating complementary approaches to achieve these improvements with greater selectivity and efficiency than before. Application of advances in structural studies and their integration with bioinformatics have provided new insights into enzyme structure- function relationships. Tools for targeting individual amino acids residues or groups of amino acids for desired changes in enzyme function have been developed by several partners and was one of the major themes of Work Package 3 led by RUG. These approaches can reduce the numbers (by as much as 2 orders of magnitude), and complexity of mutated enzyme libraries to be screened for new attributes, as. This has the impact of making huge savings in we lab resources, and offering better selectivity than random or semi random approaches to library syntheses. Introducing novel enzyme functions to existing enzyme scaffold upends up new chemistry opportunities as exemplified by the decoupling of cyclisation from protonation for the enzyme squalene hopene cyclase(SHC) by researchers at USTUTT to generate variable Brønsted-acid biocatalysts capable of operating in aqueous environments.
Innovative approaches to enzyme discovery and the description of novel families of enzymes in KYROBIO has helped to overcome freedom to operate (FTO) barriers to commercial exploitation of some biocatalysis and processes. Specific examples from the project include – the adoption of metagenomics approaches to discover new enzymes ( PROZO) and the exploration of plant sources for enzyme activities with FTO (ACIB).
Establishment of innovative metagenomics program for enzyme discovery (PROZO)
In the later stages of the project PROZO collaborated once more with CLEA in the production of novel panel of nitrilases. The EU-wide enforced Nagoya Protocol (from October 2014-onwards) imposes some limits on exploitation of material (including enzymes) derived from a foreign state’s biodiversity resources the even if the intended research is only fundamental in nature. There are many potentially wide-reaching consequences of this development, especially for small SMEs. Due to these uncertainties by simply mining the public genomics databases to find enzymes may leave the researcher exposed to future royalties, and any other retrospective actions that are now planned by the EU with respect to their current sector-focused review of Nagoya Protocol implementation. PROZO therefore to initiated in-house metagenomics screens for nitrilases using UK sourced biological samples. As a result PROZO have gained significant experience amongst commercial biocatalysis enzyme suppliers with respect to the provision of metagenomically-discovered enzyme panels. These provide enzymes that are not present in the public databases, with notable advantages, where the customer benefits from a stronger IP position and unique enzyme activities, while PROZO benefits from state-of-the-art products that are very easy to market, yet protected as they are difficult to reverse engineer. In-house metagenomics-based technologies at PROZO directly resulting from KYROBIO research in this area now include (i) a large (approx. 2.5Gb) private metagenomics database, enabling new services offerings to customers focused on unique metagenomics enzyme discovery; (ii) bespoke bioinformatics software and (iii) integration with a huge and inexpensive beyond state-of-the-art cloning projects which will have capacity to clone 10000 metagenome enzymes by the end of 2016.
CorNet, a novel module of the protein superfamily analysis system 3D- an advanced tool for the analysis of correlated mutation networks. (BPT)
Based on developments in KYROBIO BPT developed and released CorNet, a novel module of the protein superfamily analysis system 3DM. 3DM is a widely used platform in many different types of protein related research. Some examples of data in a 3DM system are - structures, sequences, structure based alignments, correlated mutation data, sequence function data, ligand binding data, mutation data from literature and other data sources, SNP data from genome sequencing projects, protein flexibility data, protein-protein interaction data, and many more.
CorNet is an advanced tool for the analysis of correlated mutation networks. Application in KYROBIO showed that correlated mutation networks extracted from superfamily alignments reflect functionally relations between residues in proteins. CorNet is hyper connected to mutation data automatically extracted from the literature for all the proteins that are in the alignment behind a CorNet network. Keywords, such as specificity, activity, enantioselectivity, etc., can be used to find if mutations related to the keyword are mostly found in the positions of a CorNet network. If a keyword scores high in a specific CorNet network, then the function that is shared by the residue positions in the network has been found. In such cases the positions in the CorNet network can be used as hotspots for protein engineering In addition to its application in KYROBIO to guide the generation of ‘smart libraries’ of , the 3DM technology has wider application in guiding protein engineering projects; drug design projects, and predicting effects of human variants in DNA diagnostics.
High-throughput molecular docking method to evaluate substrate posing and scoring; yields predictions of potential enzyme substrates and products. (BIB)
Based on developments in KYROBIO BIB released a pipeline for high-throughput docking studies. The main goal of our docking-pipeline was to create a fully automated pipeline to allow high-throughput docking. Molecular docking should help address two issues: posing (the process of determining whether a given conformation and orientation of a ligand fits the active site) and scoring (the measure of the fit of a ligand into the active site). To improve posing we used 11 docking tools (Autodock Vina, Discovery Studio, Dock6, eHITS, Gemdock, Glamdock, Glide, Gold, Plants, Surflex). Additionally, in Dock6 two different algorithms were used (Dock6Flex and Dock6Rigid) and three algorithms in Discovery Studio (CDocker, DS Ligandfit, DS Libdock). To reconcile the results from different docking software we developed a novel scoring system based on comparison of the RMSD value between each poses generated in docking experiment. With these values we generated a similarity matrix and based on this we choose the best pose. Using this approach the incorrectly predicted subsets of poses should not effect on the quality of the results.
Moreover, to provide a more accurate prediction of binding affinities we use the Prime tool to calculate the binding free energy between the receptor and ligand in its complex. We estimated the binding free energy and then re-ranked the docked conformations of the best poses of protein-ligand pairs obtained from our pipeline. The Pipeline is currently being used for predicting ligand poses in Near Attack Conformation, benchmark predicted homology models, predicting potential substrates for the reaction, predicting the main product of the reaction.
Enzyme stabilization by computational library design. (RUG)
Computational design of limonene epoxide hydrolase (LEH) with engineered stereoselectivity was achieved by application of in silico methods by researchers at RUG. Improving the efficiency of mutant library construction was demonstrated by application of bioinformatics and analysis of structural information. After in silico design and ranking of 2,500 initial multi-site mutants, a small library of 37 variants was constructed which harboured nine mutant epoxide hydrolases with the desired (R,R)- or (S,S)-product selectivity. The results suggest that a large part of the experimental screening that is common in directed evolution protocols can be replaced by in silico methods. This dramatically reduces the number of physical enzyme variants required in screening experiments providing new economies of time and materials in programs of targeted enzyme improvement. The protocol for redesign of catalytic selectivity by computation (CASCO) describes a method of work for efficient enzyme redesign, of interest to commercial organisations looking to acquire specific functional changes to biocatalysts.
Biocatalyst production and formulation
Reliable and substantial enzyme production platforms are key enabling factors to opening the way for further process development, and must be considered from the earliest stages of the evolution of those processes. Without those capacities, development of processes can be difficult, and their implementation at scale impossible. New technologies for improved protein production were developed and implemented for the biocatalysts used across KYROBIO, including optimised fermentation methodologies, and improved strains and simplified media requirements for enzyme expression. New strategies for production of biocatalyst via improved expression and fermentation protocols was the focus of Work Package 4 which was led by ACIB.
Enzyme formulations and immobilisation strategies was centred on the activities in WP 5 and was led by CLEA and concentrated on formulation for application. Enzyme immobilization is a key enabling technology for the development of industrial biotransformations, facilitating the recycling and reuse of the biocatalyst and reducing contamination of product with microbe host derived substances. Cross-Linked Enzyme Aggregates (CLEAs) consist of covalently cross-linked precipitated enzymes. CLEA has developed methods for rapid immobilisation /stabilisation of enzyme in KYROBIO which can be presented to commercial clients. Bead based solid supports for immobilisation of enzymes and other biocatalyst are familiar to many researchers and are widely used in industrial applications. However a multitude of support chemistries, porosities, bead sizes, porosities and other characteristics makes it difficult of the no specialist to decide what options to consider. (BiCT)
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High level expression strains for the intracellular and secreted production of novel hydroxynitrile lyases derived form a fern species.(ACIB VTU)
Based on its broad and highly elaborated technology platform VTU Technology generated high level Pichia expression strains in KYROBIO for the intracellular and secreted production of hydroxynitrile lyases from a fern species. The corresponding enzymes were produced for stabilization and characterisation by CLEA and ACIB partner within the KYROBIO project.
New Bacterial enzyme expression platforms, stable transformants; low cost production methods (EVO)
Bacillus subtilis production strain development was successfully conducted. The application of an operon strategy has the largest impact on expression efficiency. A combination of this technology with optimized promoters and signal peptides is very promising.
Expression systems for Squalene hopene cyclase variants (USTUTT)
USTUTT has developed and optimized the expression of the squalene hopene cyclases from Zymomonas mobilis and Alicyclobacillus acidocaldarius in E. coli. The two squalene hopene cyclases genes were cloned and expressed in the pET expression system downstream of the T7 lac promoter for tightly-regulated expression in E. coli BL21(DE3) with 0.2 mM IPTG as inducer. The active recombinant proteins obtained were around 0.4-0.8 U/mg after 4 h of induction time The enzyme produced will be used in various stereospecific C-C bond forming reactions and the possibility to apply mutants of AacSHC for synthetically important organic reactions including the Prins reaction, the bimolecular nucleophilic addition of terpenoids with alcohols and acidic isomerization of pinene monoterpenes demonstrate the opportunities of this enzyme as variable Brønsted-acid biocatalysts. The diversity of Brønsted acid-catalyzed reactions addressed with SHCs allows the catalysis of a plethora of chemical reactions having no natural counterparts. Therefore, numerous applications of SHCs can be expected, providing enzymatic access to various valuable non-natural reactions. Remarkably, nature uses this acidic machinery only for polyene cyclization of di- and triterpenes. SHC provides a potent example of the potential of enzymatic catalytic centres which are by far ‘underused’ in nature, and its exploration is a powerful method to develop new catalysts.
Robust and high performing immobilized hydroxynitrile lyases (HnLs) with freedom to operate (FTO). (CLEA ACIB)
A cooperation of CLEA with ACIB and TU Graz within KYROBIO addresses the commercial availability of robust and high performing immobilized hydroxynitrile lyases (HnLs) with freedom to operate (FTO).
Immobilised nitrile hydratase CLEA panel. (PROZO CLEA)
Combining the powerful GRASP cloning technology of Prozomix with the immobilization technology of CLEA Technologies has led to the commercial availability of a large variety of robust and recyclable enzyme preparations within KYROBIO. The production route goes from the genome to a recyclable cell free biocatalyst with high operational stability in a limited amount of steps; this is important consideration for producing panels of stable enzymes with otherwise labile activity. The success of the joint effort is the availability of an immobilised nitrile hydratase CLEA panel. These nitrile hydratase CLEAs are also available in the magnetic form and are ready for presentation to commercial customers..
Nitrile hydratase are interesting biocatalysts as they have the potential to enantioselectivity hydrate nitriles to the amides under mild reaction conditions. One possible application of stereoselective conversion using these enzymes is in the production of Levetiracetam, which is a medicine against epilepsy. C-Tech collaborated with CLEA in the early stages of the KYROBIO project by developing a conventional synthetic route for a substrate to test nitrile hydratases for the relevant activity in this synthesis.
High throughput methods to develop optimised supported transaminase catalysts BiCT)
In KYROBIO BiCT used high throughput methods to develop optimised supported transaminase catalysts by balancing formulations for all the different components for each application. BiCT’s goal is to provide very cost-effective and industrial scale biocatalyst and bioprocesses. The immobilised biocatalysts are tailored on a process specific basis, to include process specific requirements for stability, and the provision of auxiliary factors such as enzymes for regeneration of cofactors. BiCT collaborated with XZM and ING on the immobilisation of their respective transaminases for stability and reuse tests carried out by BiCT. BiCT also collaborated with C-Tech on the provision of immobilised transaminase enzymes for activity and stability tests in continuous reactors including tests in plug flow systems and electrodialysis rigs operated as biocatalysis reactors.
Tools for process analysis, process control and reaction engineering
Reaction and process engineering are critical considerations for effective technology transfer from academic laboratories to the development of operable processes in a commercial setting. Use of appropriate metrics to target process improvements for economic production will improve the efficiency of biocatalyst evaluation and subsequent process development. Similarly, an often overlooked aspect of Industrial Biotechnology R&D activity is the need to improve understanding of process intensification and control, and to design methods for efficient product recovery to deliver economic benefit from biocatalysis processes. These activities were the focus of Work Package 6 which was led by C-Tech.
Economic and environmental metrics tools evaluate enzyme catalysed process to identify specific improvements to the catalyst and process, and generate recommendations for time efficient evaluation of promising biocatalysts. (DTU)
DTU’s role in KYROBIO has been economic and environmental evaluation of other partner’s biocatalytic processes to guide further development as well as benchmarking against alternative process options. The main focus has been on production of chiral amines using amine dehydrogenase, in collaboration with X ZYM. Economic evaluation has mainly been performed using economic metrics, which are economic guidelines that can be applied at an early stage of process development and reflect the cost of different parts of the process. Depending on type of product and market size different threshold values for the metrics can be been applied. To perform evaluations at an early stage of process development can guide development to save resources and direct efforts. By applying economic and environmental metrics for evaluation to the amine dehydrogenase catalysed process in KYROBIO improvements to the catalyst and process required has been identified and the tools are recommended to be applied for time-efficient evaluation of promising biocatalysts.
Lab scale electrodialysis stack reactor design and build with applications of process control and in situ product recovery demonstrated for transaminase catalysed reaction operated at 50 ml scale. (C-Tech).
Electrodialysis (ED) is an established technique for product recovery and downstream processing in large scale commercial chemical processes, environmental remediation, and fermentation science. In KYROBIO, C-Tech collaborated with UNIGRAZ to investigate the potential for electrodialysis in the early stage research lab investigations of processes-in this case a transaminase reaction. Electrodialysis exploits differences in the mobility of chemical species in an electric fields, the presence of selective membrane compartments inside the ED stack means that species can be isolated/separated by on the basis of their charge. C-Tech’s major aim was to achieve product recovery (separation of the amine product from the reaction mixture), and to alleviate coproduct inhibition by in situ removal of the inhibitory acidic ions. Wolfgang Kroutil (UNIGRAZ) kindly supplied the enzymes used in the study, information on reaction, process methodologies, and analytical techniques. As biocatalyst, (whole cell E. coli transformants expressing Chromobacterium violaceum transaminase),was in limited supply, the test reactions were carried out at 50-100 ml volume, and a small ED stack was designed and built by C-Tech to accommodate these reaction volumes. In the selected test reaction methoxyacetone is aminated to methoxy propylamine using alanine as an amine donor; pyruvate is generated as an inhibitory coproduct. After electrodialysis 80% of the amine and 97% of the pyruvate had been removed from the reaction. 68% and 82% of the current passed had been used to transport the amine and pyruvate respectively, reflecting competition by ions derived from the whole cell biocatalyst. In addition, pH control without the use of buffer salts was demonstrated. This resulted in an increased amine yield(54%) as compared to a control, salt-buffered reaction(30%). The results are encouraging as ED can be used for process control, and to reduce the amounts of salts and other reagents used in reaction - buffer salts can be eliminated and products recovered as free acids or bases.
C-Tech also collaborated with BiCT and ING to investigate the use of immobilised transaminase enzymes in small volume ED stacks operated as semi-continuous-mode biocatalysis reactors
Based on the success of work on product separations by electrodialysis initiated in KYROBIO, C-Tech has entered into two additional collaborative R&D projects to develop lab scale electrodialysis systems which are user-friendly and suitable for scientists who are new to these techniques. Applying our expertise in electrochemical cell design C-Tech have subsequently built an integrated ED unit with unique features to allow selective removal of species from reactions and help lab based researchers get started with ED as a process development and manufacturing technology.
In addition to development of electrodialysis systems for research lab applications in biocatalysis, C-Tech also evaluated various preparations (host expression systems, preparation by C-Tech and PROZO (E. coli), and ACIB (Pichia pastoris)) of monoamine oxidase (MAON-D5, mutant previously generated at UNIMAN). The whole cell biocatalyst preparations were evaluated in a model transformation of 3-amino-3-axabicyclo [3.3.0] octane in reactor systems including stirred tank (batch), shake flask and plug flow reactors. The process benefited from measures to increase oxygen supply such as the addition of molecular oxygen to the plug flow systems, which also provide enhanced mixing. Although some progress in reducing reaction time to completion was demonstrated, especially in the plug flow reactors, it proved difficult to intensify the reactions and catalyst stability remained an issue to be addressed in future work
Potential Impact:
The Potential Impact of KYROBIO
1. Socio-economic impact
The main objective of KYROBIO is to enhance the competitiveness of the European chemical industry by substituting complex chemical syntheses using multi-step (often with precious metal catalysed reactions in hazardous chemical plants) by clean and non-polluting bioprocesses and to develop advanced engineering solutions and routes to market to enable application. Meeting this objective will lead to meeting the overarching objective of enhancing the competitiveness and sustainability of the European chemical industry by replacing complex organic synthesis by cleaner biotransformation. The development and uptake of biocatalytic processes by the chemical manufacturing industry is vital for expanding the European knowledge-based bio-economy (KBBE). KYROBIO is expected to impact significantly on the development of industrial biotechnology’s contribution to the European KBBE by increasing the use of enzymes as biocatalysts for high value chemical synthesis. In addition KYROBIO’s technical, commercial and training/educational outputs contribute to the development of Industrial Biotechnology(IB) as a Key Enabling Technology (KET) which, going forward is forecast to provide market opportunities for sustainable use of resources, wealth creation, improved lifestyle, and employment opportunities for citizens of the EU.
The development of next generation biocatalysts will allow the rapid adoption of new technologies which deliver improved performance over conventional processes, with concomitant benefits to efficiency and economy. The use of enzymatic manufacturing methodologies has the potential to ‘green’ industrial chemical production by displacing conventional chemistries which require toxic and hazardous reagents, such as metal catalysts and strong acids, and energy-intensive processes operated at high temperatures and pressures. In addition to improved economies of manufacturing, this has the potential to improve the working and living condition of those involved with, or living in proximity to chemical and process industry operations. KYROBIO helped to develop the R&D skills base of the participating scientists and technologists, increase the expertise of the partner organisations, grow an active network of collaborators, and deliver new technologies for industrial application.
The significant role of industrial participant in the project, especially the SMEs was important to setting and maintaining the focus of KYROBIO in delivering its objectives. Alongside the development of novel biocatalysts, the industrial participants of KYROBIO also played a leading role in the development and applications vital enabling technologies to allow rapid development of enzymes and biocatalysts, their integration into processes and products with commercial potential, and economic and environmental evaluation versus conventional and competing technologies.
2. Societal implications
Impact on European Research Infrastructure One of successes of KYROBIO project was to gather industrial interests and research organisations from across Europe, bringing together the necessary interdisciplinary skills in order to form a strong transnational collaboration to generate new knowledge and new technologies that will be implemented in new products and technologies. KYROBIO contributed to sustain the European research and boost collaboration of industry with universities, thus allowing both European research organizations and European industries to achieve and provide innovation and leadership in these fields. The success of the project can be judged by the impressive body of high-impact literature generated, the exploitable technical foreground knowledge generated by participants (from industry and academe), and also by a lasting legacy of enhanced cross-sector collaboration. KYROBIO placed the requirements of industry at the heart of the project, leading to the development of advanced research skills to tackle real-world problems for commercializing biocatalytic reactions. An enhanced skill base will provide benefits to the research infrastructure, but also provide vital expertise for the implementation of biocatalysis by industry.
The success of KYROBIO can also be illustrated by examples of the enhanced research infrastructure which will outlive the lifespan of the project. The joint meetings and exchanges throughout the project of young researchers for training have helped them expand their skills and knowledge of industrial biocatalysis along with the critical enabling technologies, and thereby contributed to an increasing the knowledge base in Europe. Many early stage researchers in the project have experienced collaboration with industrial project partners; presented at major international conferences (including prestigious events such as BIOTRANS 2013, BIOTRANS 20I5 and The Gordon Conferences); and have continued their association with industrial biotechnology by finding related employment in academic research and commercial organisations. This next generation of scientists and technologists will be ambassadors for industrial biotechnology and from part of the extended legacy of the KYROBIO project.
Small and medium-sized enterprises (SMEs) are the backbone of Europe’s economy, providing the majority of all new jobs. All SMEs present from the projects inception and proposal formulation stage of the project have remained active in KYROBIO until its completion. The SME participants have recorded positive outcomes from the KYROBIO project ranging from securing and expanding employment; improving their research skills; enabling new technology and product development; and expansion of their international collaborative network. This is particularly striking for early stage SMEs such as PROZO and BPT who have grown staffing levels, developed products and services (e.g. novel enzyme panels, and platform tools to access bioinformatics knowledge for enzyme discovery/improvement) as a result of work carried out in KYROBIO. New technical and commercial opportunities have been identified by SME participants working in collaboration. For example, CLEA and PROZOMIX have worked together to provide a new product for their joint benefit - enzymes produced by PROZO, formulated as stabilised immobilised preparations by CLEA. C-Tech’s involvement, both as coordinator and by its technical role in KYROBIO, increased its exposure to players in the area of industrial biotechnology and opened new market opportunities for its engineering design and build capabilities. Commercially relevant outcomes such as these derived from KYROBIO strengthen the SME participants’ capability to continue to offer high value employment and commercial growth in the KBBE sector.
Continuing research collaborations have been strengthened and established, for instance, a number of KYROBIO partners (UNIMAN, USTUTT, DTU, CLEA, BiCT PROZO C-Tech and BPT) form part of the of the consortium of the FP7 project BIOOX(613849). Such examples of sustained cooperation can be found across the consortium, were individual participants have gone on to develop additional co-funded and commercial collaborations for further exploitation of concepts established in KYROBIO.
3. The main dissemination activities
The supradisciplinary approach taken in KYROBIO encourages researchers in industrial biotechnology to gain understanding and technical competence in the emerging techniques and tools for topics such as enzyme engineering and enzyme production. To this end several hands-on training courses, web-based informatics tools, webinars and other dissemination and training activities were organised as part of KYROBIO. The lab courses will be reprised post project.
Training courses. Courses in computation-supported enzyme engineering, now to be offered to PhD students and post docs on a biannual basis.(RUG) A KYROBIO masterclass held in Groningen in April 2015 was aimed to make students and post-docs familiar with the use of computational and bioinformatics tools for enzyme engineering. The improved accessibility of many tools brings them within reach of the wet biochemistry and organic chemistry labs. The program consisted of morning lectures by various speakers on basic principles of computational methods and recent highlights from their own research. Speakers from Industry and Academic backgrounds were affiliated to KYROBIO, or similar projects (BE-Basic, MicroB3, P4FIFTY) aimed at biocatalyst development.
Hands-on Pichia Lab course: "From Idea to Protein Production" was offered to academics and industrial researchers (ACIB). ACIB have provided training and knowledge transfer in practical methods for protein production, one of the key enabling technologies for biocatalyst development and application, helping to address the apparent skills shortage within this specialised area. The yeast Pichia is a useful research and industrial scale host for biocatalyst production, but remains unfamiliar to many researchers in this field.
Publications in peer reviewed journals (USTUTT, UNIGRAZ, RUG, UNIMAN, DTU, and ACIB); Webinar on Squalene Hopene Cyclases (USTUTT); Webinar highlighting KYROBIO technical achievements (KTN LTD). As could be anticipated by the high quality research output generated by the top ranking research group research organisations and universities, original research was published in 28 articles in peer reviewed journals. Further peer reviewed publications are in preparation by several KYROBIO participants. In addition, the research output afforded many opportunities for project dissemination to the wider research community at numerous international conferences. Two public webinars summarising research achievements were delivered during the project and recordings have been made available on social media and various public websites.
Presentations to civil society, including events for schoolchildren, Museum exhibits, Science Fairs and Open Day (UNIMAN). KYROBIO researchers at the University of Manchester have been heavily involved in outreach initiatives to raise awareness and widen participation in the area if industrial biotechnology. Engagement was with schools across the north of England, at the annual national science and engineering week, British science week and Manchester Institute of Biotechnology open day events. There has also been involvement in local community engagement initiatives such as Manchester science week, faculty open days and museum exhibitions. KYROBIO has also been represented at national outreach events such as the Royal Society Summer Science Exhibition 2013 and Great British Bioscience Festival 2014. Engagement activities have ranged from hands-on practical experiments and equipment demonstrations to lab tours and research talks.
International networking event, Public website and 7 project newsletters (KTN LTD). A major dissemination event was held in Brussels on 3rd Dec 2013 in conjunction with the BIONEXGEN project (266025). At the event, attended by an international audience from academia, industry and policymakers, project results were disseminated by talks; a multimedia exhibition was provided by project participants; and a panel discussion with representatives of industry and academia promoted the benefits of collaborative research in the development of Europe’s capability in Industrial Biotechnology. A publicly accessible website was established at the project commencement with information on the project and participants, news updates on project activities and general interest articles on the themes of industrial biotechnology. News and updates on project progress were disseminated to interested parties by a regular series of newsletters circulated by email and posted on the project website.
Dissemination to the widest possible audience of scientific specialists, industrial end-users, policy makers, but importantly the European public, has been at the heart of KYROBIO. By participating in outreach activities, including open days for schools, public science fairs, and engaging with diverse media (including specialist, academic trade and general press), KYROBIO researchers have helped to increase public awareness of EU research and industrial biotechnology (IB). KYROBIO researchers has been involved in the coordination and delivery of a Massive Open Online Course (MOOC) in Industrial Biotechnology as part of the University of Manchester’s research beacons initiative. The course, which is due to be released worldwide to thousands of participants during the 2016, is being designed to introduce students from diverse backgrounds to the principles of industrial biotechnology, with a focus on enzyme technology and biocatalysis for the production of pharmaceutical compounds. Submodules of the MOOC are being delivered by both internal and external academic and industrial partners, including members of KYROBIO team.
By contributing to the public understanding of IB, this project will help to improve consumer acceptance of biocatalytic manufacturing for improved functionality, safety, and environmental credentials of IB products. The enthusiastic interaction of KYROBIO scientists with young people and educators will help to increase the uptake of STEM subjects (Science, Technology, Engineering and Mathematics) by school children and students progressing to higher education, in order to nurture future generations of researchers, engineers, biocatalyst-end users, and the wider European chemical and process industries.
4. Exploitable results
New C-C and C-N bond forming biocatalysis reactions demonstrated and developed within the KYROBIO project have extended the range of chiral molecules which can be synthesised in this manner. KYROBIO has delivered new technologies for the synthesis of industrially important molecules including chiral amines, terpenoids and chiral cyanohydrins. These molecules are amongst the most important building blocks for agrochemicals, fine-chemicals and pharmaceuticals. Chirality is a critical feature in biological activity and functionality in each case. Conventional technologies for the synthetic production of chiral molecules are often wasteful and require harsh reaction conditions. Chemical routes to chiral amines can be particularly challenging, and many molecules remain completely inaccessible. Traditional manufacturing methodology for the production of chiral chemicals often require deracemisation of products, therefore limiting yields to a maximum 50%, since the unwanted enantiomer is not required.
As part of the biotechnology toolkit these enzymes play the central role in generating new products and processes. The project met the overarching challenge in this area by control of reaction stereochemistry to add value to KYROBIO chemistry. Excitingly, in many cases these new technologies, including the use of enzyme cascades in some instances, also allow access to a wider range of substrates, to generate completely novel products and processes that were not previously realisable by biocatalysis. This has the potential to not only change routes for chemical manufacture, but also to introduce new products and new functionalities in high value chemical products.
The reengineering of enzymes to generate new activities and uncoupled enzyme activities are exemplified in several instances notably in the modification off SHC enzyme by USTUTT. and generation of Amine dehydrogenase activity XZM-JM. Amine dehydrogenases can be can be generated from amino acid dehydrogenases by mutating 2 or more residues in leucine dehydrogenases. The new enzymes can supply amination of commercially interesting ketones and represent a n expansion of enzyme toolkit as an interesting complement /alternative to the use of transaminases.
The mechanism guided engineering approach of the squalene hopene cylase AacSHC will allow to gain a greater fundamental understanding in discovering new enzyme activities in the lab. By generating enzyme catalysts of Brønsted acid reactions will facilitate the synthesis of new product molecules of high added value
Applications for the new biocatalysts can be envisaged in drug- agrochemical- and speciality chemical manufactures.
• Engineered amine dehydrogenases (AmDHs) available as part of screening kit. (XZM-JM)
• Novel Transaminase variants with new substrate range available as kit and for further development (XZM-JM)
• Application of bacterial Ammonia Lyase (EncP) and its variants to produce unnatural amino acids. (UNIMAN)
• Novel terpenoid products of industrial interest produced by application of Squalene hopene cyclases (SHC) to an extended substrate range. (USTUTT)
• Decoupling of polycyclization chemistry from Alicyclobacillus acidocaldarius SHC to yield a platform for enzymatic Brønsted acid catalysis in water. (USTUTT)
• Enzyme cascade combining ω-Transaminases and strictosidine synthases to yield high added-value alkaloid derivatives with additional chiral centre. (UNIGRAZ)
• Expansion of activity and substrate range of Granulicella tundricola HNL(hydroxynitrile lyase with a cupin fold) to the production of chiral cyanohydrins, important intermediates in pharmaceutical manufacturing (ACIB)
• Novel hydroxynitrile lyases cloned and expressed by reverse genetics approach from fern transcriptome (Davallia teyermanii). (R)-cyanohydrins are produced in a highly selective manner. (ACIB)
Tools for enzyme discovery and modification, frequently achieved by development and application of structural and bioinformatics methodologies, was an important part if KYROBIO’s aim to improve the biocatalysis (product) repertoire available for enzyme systems. KYROBIO has produced toolboxes for preparation of modified biocatalysts by generating complementary approaches to achieve these improvements with greater selectivity and efficiency than before. Innovative approaches to enzyme discovery and the description of novel families of enzymes in KYROBIO has helped to overcome freedom to operate (FTO) barriers to commercial exploitation of some biocatalysis and processes. Specific examples from the project include -
• Establishment of innovative metagenomics program for enzyme discovery (PROZO)
• CorNet, a novel module of the protein superfamily analysis system 3DM- an advanced tool for the analysis of correlated mutation networks. (BPT)
• High-throughput molecular docking method to evaluate substrate posing and scoring; yields predictions of potential enzyme substrates and products. (BIB)
• Efficient enzyme modification protocols by computational library design. (RUG)
Reliable and substantial enzyme production platforms are key enabling factors to opening the way for further process development, and must be considered from the earliest stages of the evolution of those processes. Examples of improved/exploitable microbial enzyme production systems were demonstrated in KYROBIO including productions of plant derived HNL enzyme in Pichia pastoris (VTU); methanol-free enzyme expression in Pichia pastoris (ACIB); and stable chromosomal based enzyme expression and secretion in Bacillus (EVO). Improved enzyme production systems have multiple positive economic and environmental impacts. These improvements in enzyme production could production costs by reducing materials and energy usage. Moving away from promotor systems which are dependent on inducer compounds (methanol in the case of Pichia) and selection systems which require the presence of antibiotics reduce both material cost and environmental burden of enzyme production. Specific examples of exploitable KYROBIO outputs in this area include -
• High level expression strains (Pichia) for the intracellular and secreted production of novel hydroxynitrile lyases derived form a fern species.(VTU)
• New Bacillus host and expression systems for stable enzyme expression and secretion of enzymes.
Enzyme immobilization is a key enabling technology for the development of industrial biotransformations, facilitating the recycling and reuse of the biocatalyst, as well as offering improvements in catalyst stability. Cross-linked Enzyme Aggregates (CLEAs) consist of covalently cross-linked precipitated enzymes. CLEA has developed methods for rapid immobilisation /stabilisation of enzyme in KYROBIO which can be presented to commercial clients. Bead-based solid supports for the immobilisations of enzymes are available in many formats varying in many aspects including diameter, porosity, stability and method of linkage. Tailoring the immobilisation methodology to the process application needs has been undertaken by scientists at BiCT to produce a suite of immobilised transaminase enzymes developed within KYROBIO.
• Robust and high performing immobilized hydroxynitrile lyases (HnLs) with freedom to operate (FTO). (CLEA ACIB)
• Immobilised nitrile hydratase CLEA panel. (PROZO CLEA)
• High throughput methods to develop optimised supported transaminase catalysts by balancing formulations for all the different components for each application. (BICT)
Process engineering and tools for process design are critical considerations for effective technology transfer from academic laboratories to the development of operable processes in a commercial setting. Use of appropriate metrics to target process improvements for economic production will improve the efficiency of biocatalyst evaluation and subsequent process development. Similarly, an often overlooked aspect of IB R&D activity is the need to improve understanding of process intensification and control, and to design methods for efficient product recovery to deliver economic benefit from biocatalysis processes. Specific examples of exploitable KYROBIO outputs in this area include -
• Economic and environmental metrics tools evaluate enzyme catalysed process to identify specific improvements to the catalyst and process, and generate recommendations for time efficient evaluation of promising biocatalysts. (DTU).
• Lab scale electrodialysis stack reactor design and build with applications of process control and in situ product recovery demonstrated for transaminase catalysed reaction operated at 50 ml scale. (C-Tech)
List of Websites:
www.kyrobio.eu