WG3: Experimental evaluation and characterization
Lígia O. Martins
NOVA University of Lisbon, Portugal
University of Stuttgart, Germany
Mutant libraries preparation and screening – The predicted mutant libraries from WG1 and 2 will be created within WG3. Open source tools such as AAscan or Primer X will be used to automatedly design the mutagenic PCR primers for site-directed mutagenesis. For a typical library size of a few hundred mutants, a reliable primer design strategy and a high-PCR success rate are critical. Transformation and expression will be performed in microtiter or deep-wells plates using multichannel pipettes or liquid handling robots. Efficient enzyme purification will be enabled by expressing His-tagged proteins and using paramagnetic precharged nickel particles. High-throughput sensitive assays will allow to determine thermostability and activity profiles. Activity-based microfluidic assays will be used for enzymes converting hydrophobic compounds using a capillary-based droplet microfluidic platform. The implementation of these miniaturized approaches, allow parallel (and multiple) 96 gene expressions, protein purifications, and enzymes assays to be performed in 1 week time. The strategy also provides good reproducibility upon scale-up as the same culture and purification conditions are used. Library screening and subsequent characterization of the best hits requires production in a microbial host at sufficient quantities. To ascertain high-level production of all COZYME target enzymes, codon optimized synthetic genes encoding these enzyme variants will be used. This circumvents laborious molecular cloning, we will take advantage of the in-house developed tools of the partners that participatein WG3. All enzyme variants will initially be produced in Escherichia coli or in yeast if required. However, microbial expression is often not straightforward and therefore an advanced workflow for mutant screening based on in vitro expression and subsequent monitoring of enzyme activity will be developed which would allow a more rapid protocol for expression and screening, eliminating the need for transformations. Importantly, all WG3 partners have the facilities and expertise to carry out expression and purification trials of the improved COZYME target enzymes in a range from small scale (multiwell-plate
format) to 2L fermentation and required DSP steps. Biochemical and biophysical characterization – Various biochemical and biophysical techniques will be employed to assess the relationship between sequence, function and structure of hit variants obtained from the HTS screenings. The soluble expression of enzyme variants will be measured by gel band
densitometry. Thermodynamic stability will be assessed by fluorescence emission or differential scanning calorimetry allowing to determine the melting temperature of variants. Deactivation kinetics or long-term stability will be assessed the half-life of enzyme at optimal temperature reaction conditions. The characterization by steady-state and stopped-flow kinetics will provide information on the variant enzyme’s specificity and mechanisms. Different spectroscopic techniques (UV-Vis, EPR, cyclic voltammetry) will allow the characterization of their redox centers and spectral features. Protein crystallography will be used for obtaining structural details at atomic level. The elucidation of 3D structures of wild-type and engineered enzymes is paramount for the achievement of the COZYME objectives. Molecular determinants and key residue positions for improved properties will be identified using molecular dynamics (MD), docking and quantum-based methods. While MD will help to elucidate
important conformational shifts due to mutations, docking will show differences in substrates migration and positioning at the active site, QM/MM will pinpoint the electronic effects that are responsible for
altered activity (with respect to the wild type enzymes). The functional and structural characterization of enzyme variants will allow to (1) map and understand the improved fitness onto properties of proteins by establishing genotype-phenotype relationships, (2) advance our understanding of structure-function determinants and mechanism of enzyme specificity and stability, (3) generate optimized biocatalysts (4)
start new rational or semi-rational enzyme design cycles.
An infrastructure based on the standardized data exchange format EnzymeML will be implemented to enable reporting, exchange, and storage of enzymatic data according to the FAIR data principles. The
research data management infrastructure guarantee reproducibility of experimental data and enable the integration of large-scale experimental data with results from advanced modelling methods such as simulation and machine learning. An EnzymeML Application Programming Interface integrates electronic lab books with modelling platforms and databases on enzymatic reactions. The research data management infrastructure enables the seamless flow of data between measurement and modelling,
and thus contributes to the vision of a unified research data infrastructure for catalysis research.
The main objectives of WG3 are to: (1) setup medium-throughput experimental pipeline protocols for generating focused mutant libraries and activity/stabilty screening assays; (2) develop robust and scalable protocols for the recombinant expression of improved COZYME target enzymes in different microbial production hosts; (3) characterize the best variants using a combination of techniques (kinetic,spectroscopic, stability) and structural (X-ray, cryoEM, molecular dynamics); (4) advance our understanding of enzyme catalysis through elucidating structure-activity relationships and advance our predictive ability for efficient engineering of novel enzymes for new applications; (5) establish a research data management infrastructure based on the F.A.I.R data principles for the seamless integration of experimental and computational data.
Specific Tasks of WG3 are:
Construction of the predicted mutant libraries.
Development of the medium-throughput screening technology.
Experimental screening of the predicted mutant libraries and biochemical characterization of the best mutant variants.
Efficient production and structural characterization of best mutant variants.
Apply a joint research data management infrastructure and demonstrate its feasibility and usefulness.