Enzyme Fusion & Oligomerization Service

Enzyme Fusion & Oligomerization is a strategic protein engineering service focused on designing multi-functional enzymes or organizing enzymes into defined supramolecular complexes. Enzyme fusion involves genetically linking two or more catalytic domains via a designed linker to create a single polypeptide chain, often forming synthetic metabolic pathways (metabolons) that increase local substrate concentration and enhance flux. Oligomerization engineering focuses on controlling the assembly state (e.g., dimer, tetramer) or inducing hetero-oligomer formation to introduce allosteric regulation or stabilize the protein scaffold.

CD Biosynsis offers comprehensive CRO services for designing and validating engineered enzyme assemblies. Our platform uses computational modeling to design optimal linker sequences for fusion constructs, ensuring domain flexibility and avoiding steric clashes. For oligomerization, we utilize interface engineering to create specific, strong, and directional protein-protein interactions. This approach is essential for enhancing the efficiency of multi-step cascade reactions, creating self-assembling enzyme nanoparticles, and engineering novel forms of regulatory control within synthetic biological systems.

Highlights

We provide tools to structurally organize enzymes, enhancing efficiency and enabling complex regulation.

• Enhanced Metabolic Flux: Fusion of sequential pathway enzymes to create substrate channeling, eliminating intermediate diffusion bottlenecks and accelerating the overall cascade rate.
• Optimized Linker Design: Computational modeling to select the optimal linker length, sequence, and flexibility to maintain the native activity of each fused domain.
• Controlled Assembly State: Rational engineering of protein-protein interfaces to enforce a desired oligomeric state (e.g., forcing a monomer into a functional dimer or tetramer).
• Creation of Synthetic Metabolons: Designing scaffolds or fusion proteins that mimic natural metabolons, achieving high local concentrations of intermediates.

Applications

Oligomerization and fusion are key strategies in metabolic engineering and material science:

Metabolic Pathway Engineering

Creating highly efficient, encapsulated biosynthetic pathways for the overproduction of high-value chemicals, fuels, or pharmaceuticals.

Enzyme Nanoparticle Biocatalysis

Designing self-assembling enzyme complexes for easy separation, recovery, and reuse in industrial reactors.

Conditional Stability and Regulation

Engineering enzymes to be active only in a specific oligomeric state, allowing regulation via concentration or small-molecule induced assembly.

Multi-Enzyme Diagnostics

Creating genetically fused enzyme reporters for simultaneous detection of multiple analytes in a single assay system.

Platform

Our platform combines computational design with biophysical validation methods to control protein organization.

Linker Modeling and Optimization

Using Molecular Dynamics (MD) to screen and optimize flexible (Gly-Ser) or rigid (Pro-rich) linkers to ensure maximal domain mobility and minimal interference.

Interface Redesign for Oligomerization

Rational mutagenesis of surface residues to create new hydrophobic patches or electrostatic interactions that drive specific self-assembly into dimers, trimers, etc.

Computational Docking of Fusion Domains

Modeling the interaction of the fused domains and linkers to ensure the active sites are spatially oriented for efficient substrate channeling.

Biophysical Validation (SEC-MALS, AUC)

Experimental confirmation of the designed oligomeric state using Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) or Analytical Ultracentrifugation (AUC).

Cascade Reaction Kinetics

Quantifying the overall rate enhancement of the fused multi-enzyme system compared to the non-fused mixture, verifying the channeling effect.

Workflow

Our Enzyme Fusion and Oligomerization workflow is designed to ensure stable assembly and optimal function:

• Enzyme Selection and Structural Modeling: Select the enzymes (domains) and obtain/model their 3D structures. Determine the required orientation for substrate channeling.
• Fusion Linker Design: Design and computationally screen a set of linker lengths and compositions (e.g., GGGGS repeats) to achieve the ideal spatial distance and flexibility between the domains.
• Oligomerization Interface Design (if needed): Identify or redesign surface residues to create highly specific, non-native interfaces that drive the formation of the desired oligomer (e.g., dimer).
• Gene Construction and Synthesis: Create the final gene construct, including the optimized linker and any oligomerization mutations. Synthesize and clone the DNA.
• Expression, Purification, and Biophysical Validation: Express the construct and purify the protein. Use SEC-MALS/AUC to rigorously confirm the designed molecular weight and oligomeric state.
• Functional and Kinetic Analysis: Perform cascade assays to quantify the overall catalytic efficiency, demonstrating the successful achievement of substrate channeling or enhanced stability.

CD Biosynsis delivers structured enzyme systems with verified assembly and enhanced cascade performance. Every project includes:

• Optimized Gene Sequence: The final sequence of the fusion protein, including the verified linker and any oligomerization mutations.
• Biophysical Validation Data: SEC-MALS or AUC data confirming the designed oligomeric state and structural integrity.
• Cascade Enhancement Metrics: Quantitative comparison of the catalytic rate (Vmax) of the fused/oligomerized system vs. the unlinked, non-assembled enzymes.
• 3D Model of Assembly: The computational model showing the spatial arrangement of the linked domains or the oligomerized units.