Structure-based enzyme engineering for strain optimization

Overview

Innophore supports strain optimization through structure-based enzyme engineering for microbial production systems. The approach focuses on improving pathway-relevant redox and hydrolytic enzymes to increase product formation, enhance process stability, and reduce undesired side products.

The methodology combines Innophore’s Catalophore™ technology with evolutionary conservation analysis, rational mutagenesis, computational protein design, ancestral sequence reconstruction, and deep learning-guided predictions. These methods enable the prioritisation of enzyme variants that support efficient and selective biosynthetic pathways.

Technical Approach

• Catalophore™ analysis: Compares enzyme binding sites and active-site environments based on 3D shape and physicochemical properties.
• Pathway-relevant enzyme analysis: Identifies catalytic features that may influence pathway performance and product formation.
• Evolutionary conservation analysis: Highlights residues associated with activity, structural integrity, substrate recognition, or stability.
• Rational mutagenesis: Prioritises targeted amino-acid substitutions based on structural and functional information.
• Computational protein design: Uses tools such as Rosetta and ProteinMPNN to design and evaluate enzyme variants.
• Ancestral sequence reconstruction: Explores sequence templates with potentially useful properties for enzyme optimisation.
• Deep learning-guided predictions: Supports the prioritisation of sequence and structure designs.
• Structure-based design campaigns: Target stability-enhancing motifs and residues relevant to selectivity and catalytic performance.

Strain Optimization Focus

The workflow supports strain optimization by improving enzymes within microbial production pathways. Depending on the project, the focus may include:

• Increasing enzyme activity and product conversion
• Improving product selectivity
• Reducing undesired side products
• Enhancing enzyme stability under process conditions
• Improving substrate recognition and catalytic performance
• Selecting suitable enzyme variants for pathway integration
• Optimising redox and hydrolytic steps in biosynthetic routes

In previous enzyme engineering projects, redox enzyme variants demonstrated increased product conversion and selectivity, together with reduced side-product formation. Hydrolytic enzyme variants showed improvements in activity and stability. These results illustrate how structure-informed enzyme optimisation can contribute to microbial production systems.

Partnership Opportunities

Innophore can support projects involving:

• Computational enzyme optimisation for microbial strains
• Improvement of pathway-relevant redox enzymes
• Engineering of hydrolytic enzymes for biocatalytic processes
• Reduction of side-product formation in biosynthetic pathways
• Selection and prioritisation of enzyme variants
• Structure-guided improvement of pathway efficiency
• Adaptation of the workflow to different enzyme classes and production systems

The workflow generates prioritised enzyme designs for experimental evaluation. Final strain and enzyme performance should be confirmed through biochemical, fermentation-based, and process-relevant assays.