Periodic Reporting for period 1 - TreasureDrop (Directed Evolution of Enzyme for Applied Biocatalysis at Ultrahigh Throughput in Picoliter Droplets)
Periodo di rendicontazione: 2017-05-01 al 2019-04-30
Enzymes have established as a new class of catalysts, but remain insufficient in terms of process compatibility, activity or selectivity. Enzyme engineering – i.e. directed evolution or semi-rational protein design – is currently the most promising approaches to improve or alter known enzymes. Further, the field of metagenomics bears high potential to find new scaffolds. In either case, the detection of enzymatic activity via an optical read-out is a simple, fast and versatile tool to find desired biocatalyst. However, a throughput of ≤105samples/day using plate formats is limiting the potential of these assays. Droplet-based single-cell assays boost the chances to succeed by enabling ultra-high throughput (uHTP) screening of ≥107samples/day – if considering that success often correlates with numbers. We aimed at using this technique to showcase a proof-of-principal enzyme engineering campaign targeting an industrially relevant application. In addition, its potential for functional metagenomics was investigated.
In conclusion we are convinced that the main objectives of the action were not just reached, but even exceeded based on the results obtained during the last 2 years. This was possible due to an outstanding support from all project participants and access to the newly established robotic facilities at JM. The microfluidic assay technology has been confirmed as a highly promising technique to not only access better, but also new enzymes for industrial applications. The action was recently selected by the Innovation Radar and its exploitation as a service to customers is currently being discussed at JM. Even after the end of the action, JM and the university are in close contact (including me advising students) and are discussing future collaboration opportunities.
In conclusion we are convinced that the main objectives of the action were not just reached, but even exceeded based on the results obtained during the last 2 years. This was possible due to an outstanding support from all project participants and access to the newly established robotic facilities at JM. The microfluidic assay technology has been confirmed as a highly promising technique to not only access better, but also new enzymes for industrial applications. The action was recently selected by the Innovation Radar and its exploitation as a service to customers is currently being discussed at JM. Even after the end of the action, JM and the university are in close contact (including me advising students) and are discussing future collaboration opportunities.
The enantioselective reduction of tetrahydrothiophene-3-one by an alcohol dehydrogenase (ADH) was targeted as it yields an important API (active pharmaceutical ingredient) building block. A set of mutant libraries was generated based on random mutagenesis, random deletions, and semi-rational simultaneous mutagenesis of 3-6 amino acids to study different evolutionary pathways. The best variants were identified using a previously established assay upon addition of required adjustments and validating control experiments. Testing of 2*106 mutants resulted in 3.000 promising hits, which were further narrowed down using conventional screening techniques to give 200 final hits with improved catalytic properties compared to the wild-type (WT) under the applied assay conditions. We were pleased to find that 20% of those hits also showed improvement under industrial reaction conditions. Our results highlight that – regardless of compromises required for uHTP applications – the used technique allows identification of industrially relevant enzymes. Fortunately, the semi-rationally designed mutants and those obtained through random mutagenesis showed comparable improvements, which makes our approach broadly applicable to different goals. A random combination of identified beneficial mutations gave a second set of mutant libraries, which are currently under investigation. We expect this experiment to give a highly improved biocatalyst after only 2 rounds of evolution; compared to traditional strategies which require up to 20 rounds. In collaboration with a PhD student in the Hollfelder lab, the crystal structure of the wild-type enzyme was solved and analytics for a detailed characterization of the enzyme kinetics have been established; both will be applied to explain the impact of identified mutations. The obtained data provides a unique basis to investigate epistatic interactions and their – usually neglected - importance within enzyme engineering campaigns. Remaining experiments to finish the project are currently being performed under my supervision.
The initially proposed project turned out to be a ‘text book’ example for successful project management, which allowed time to go a step further and investigate how the field of functional metagenomics could benefit from microfluidic assays. In line with the main objective, our efforts focused at the identification of novel ADHs. Regardless of the initially highlighted potential of colorimetric assay for this purpose, the direct transfer of known applications into the microfluidic format is hampered by the unique system requirements. We have identified a highly water-soluble fluorescent dye as a suitable label for fluorogenic substrates for droplet-based applications. Constituting an example, a set of substrates was synthesized and can be expanded to many different enzyme classes in the future. Assay evaluation was performed using a positive control obtained from the JM enzyme collection. Additional studies indicated that the assay can give access to enzymes from different enzyme families without sequence bias (only 20% sequence identity between hits within the JM enzyme collection) and thus, is likely to allow identification of biocatalysts within new sequence space. To our satisfaction, the generation and screening of a metagenomic soil library (sample taken just outside the JM laboratories in Cambridge) resulted in the identification of a new ADH. Most similar proteins in the database show around 70% sequence identity and are mainly annotated as amidases with a minor amount expected to be oxidoreductases, which potentially suggests a novel enzyme family or reaction mechanism. More detailed investigations are required and will be performed by the Hollfelder group. Further efforts to generate additional (meta)genomic libraries to identify further biocatalysts are ongoing.
Research is still ongoing to raise the impact of the envisaged publications; however, we believe that the obtained results will lead to ≥2 publications and reveal general principles helping microfluidics assays to flourish in the future. In addition, this work significantly extends targetable goals using microfluidics, which will be highlighted in a review providing a complete overview of biacatlyst-related droplet screens to date.
The initially proposed project turned out to be a ‘text book’ example for successful project management, which allowed time to go a step further and investigate how the field of functional metagenomics could benefit from microfluidic assays. In line with the main objective, our efforts focused at the identification of novel ADHs. Regardless of the initially highlighted potential of colorimetric assay for this purpose, the direct transfer of known applications into the microfluidic format is hampered by the unique system requirements. We have identified a highly water-soluble fluorescent dye as a suitable label for fluorogenic substrates for droplet-based applications. Constituting an example, a set of substrates was synthesized and can be expanded to many different enzyme classes in the future. Assay evaluation was performed using a positive control obtained from the JM enzyme collection. Additional studies indicated that the assay can give access to enzymes from different enzyme families without sequence bias (only 20% sequence identity between hits within the JM enzyme collection) and thus, is likely to allow identification of biocatalysts within new sequence space. To our satisfaction, the generation and screening of a metagenomic soil library (sample taken just outside the JM laboratories in Cambridge) resulted in the identification of a new ADH. Most similar proteins in the database show around 70% sequence identity and are mainly annotated as amidases with a minor amount expected to be oxidoreductases, which potentially suggests a novel enzyme family or reaction mechanism. More detailed investigations are required and will be performed by the Hollfelder group. Further efforts to generate additional (meta)genomic libraries to identify further biocatalysts are ongoing.
Research is still ongoing to raise the impact of the envisaged publications; however, we believe that the obtained results will lead to ≥2 publications and reveal general principles helping microfluidics assays to flourish in the future. In addition, this work significantly extends targetable goals using microfluidics, which will be highlighted in a review providing a complete overview of biacatlyst-related droplet screens to date.
Experiments within the protein engineering projects including kinetic enzyme characterization, epistatic studies, evaluating the 2. set of libraries and preparative scale syntheses will be completed by a PhD student in the Hollfelder lab under my supervision. With regard to the functional metagenomics, generation of additional (meta)genomic libraries and identification of further biocatalysts is envisaged. Presentations of this work at conferences and meeting have already resulted in 2 international collaborations and given me the opportunity to gain experience is team leading and project management.
Considering the ongoing efforts to extend the Nagoya Protocol to gene sequence information, enzyme engineering might gain even more importance soon. The microfluidic droplet-screening - besides of its unprecedented throughput - is more cost- and resource-efficient compared to available alternatives which is in agreement with green chemistry ideas. We are convinced that showing its potential in an industrial setting will bring this technology closer to state-of-the-art providing the European community a significant advantage for the future of biotechnology.
Considering the ongoing efforts to extend the Nagoya Protocol to gene sequence information, enzyme engineering might gain even more importance soon. The microfluidic droplet-screening - besides of its unprecedented throughput - is more cost- and resource-efficient compared to available alternatives which is in agreement with green chemistry ideas. We are convinced that showing its potential in an industrial setting will bring this technology closer to state-of-the-art providing the European community a significant advantage for the future of biotechnology.