Enzyme Cascade Design Service

Enzyme Cascade Design, or Multi-Enzyme Cascade (MEC) reaction design, involves assembling a sequence of two or more distinct enzyme-catalyzed reactions into a single reaction vessel or engineered cell. The product of the first enzyme becomes the substrate for the second, and so on, creating an efficient and modular biocatalytic pathway that transforms a simple starting material into a complex product. This approach eliminates the need for isolating intermediates, minimizes byproducts, and often enables reactions that are difficult or impossible using traditional chemical synthesis, particularly in the fields of asymmetric synthesis and natural product production.

CD Biosynsis offers expert services in Enzyme Cascade Design, covering the entire process from enzyme selection and engineering to modular assembly and optimization. We specialize in designing robust cascades that address key challenges such as enzyme compatibility, cofactor regeneration, and substrate channeling. Our services include optimizing enzyme ratios, engineering individual enzymes for enhanced compatibility, and utilizing advanced spatial organization techniques, such as co-immobilization or compartmentalization, to maximize reaction flux and yield. This results in highly efficient, environmentally friendly, and scalable production methods for complex molecules.

Highlights

Enzyme Cascade Design delivers streamlined and efficient synthetic routes for complex molecules.

• Enhanced Efficiency: Eliminates intermediate purification steps, leading to higher overall reaction yields and reduced processing time.
• Cofactor Regeneration: Cascades can be designed to include an efficient enzyme system for the in situ recycling of expensive cofactors (e.g., NADH, ATP).
• Substrate Channeling: Physical proximity of enzymes can increase the local concentration of intermediates, accelerating reaction kinetics and minimizing byproduct formation.
• Mild Conditions: All reactions occur under mild, aqueous conditions (ambient temperature and pH), reducing energy input and minimizing side reactions.

Applications

Enzyme cascades are pivotal in creating complex, high-value molecules and sustainable synthesis:

Stereoselective Synthesis

Combining multiple steps, often redox and ligation, to synthesize chiral compounds with high enantiomeric and diastereomeric purity in a one-pot reaction.

Bioproduction of Natural Products

Reconstituting complex biosynthetic pathways in vitro or in engineered microbial hosts for the efficient synthesis of pharmaceuticals and bioactive compounds.

Sustainable Chemical Manufacturing

Developing green chemistry routes that use inexpensive, bio-derived feedstocks (e.g., sugars) to produce industrial chemicals and biofuels.

Cofactor Regeneration Systems

Designing custom cascades that include a dedicated enzyme (e.g., a dehydrogenase) to continuously recycle high-cost cofactors like ATP or NADP(H).

Platform

Our platform combines bioinformatics, modular assembly, and advanced immobilization techniques for cascade optimization.

Enzyme Compatibility Profiling

Bioinformatic analysis and experimental testing to select enzymes that function optimally under shared conditions (pH, temperature, ionic strength).

Modular Gene Assembly

Use of standardized cloning methods (e.g., Golden Gate) to assemble multiple enzyme genes onto a single plasmid or chromosome for co-expression.

Co-Immobilization for Channeling

Physical linking of sequential enzymes onto an inert support (beads, matrix) to achieve spatial organization and enhance mass transfer of intermediates.

Tuning Enzyme Ratios

Experimental optimization of the relative concentrations or expression levels of each enzyme to prevent intermediate accumulation and bottlenecks.

In Vivo or In Vitro Setup

Ability to implement the final cascade using either purified enzymes (in vitro) or whole, engineered microbial cells (in vivo) for biocatalysis.

Workflow

Our Enzyme Cascade Design follows a structured process to ensure functional and robust multi-step synthesis:

• Pathway Mapping and Enzyme Selection: Define the desired multi-step conversion. Select robust enzymes for each step and the regeneration system.
• Individual Enzyme Optimization: Engineer each selected enzyme for desired properties (e.g., stability, activity, substrate range) using directed evolution if necessary.
• Compatibility and Expression Tuning: Verify that all enzymes are active under the same conditions. Adjust gene expression levels or purified enzyme ratios for maximum flux.
• Cascade Assembly and Immobilization: Assemble the genes into a single host or co-immobilize the purified enzymes onto a carrier for spatial optimization.
• Proof-of-Concept Reaction: Perform the one-pot/whole-cell cascade reaction and quantify the final product yield and purity using chromatography.
• Process Optimization: Adjust process parameters (pH, temperature, buffer, enzyme loading) and perform final kinetic modeling to maximize production and stability.

CD Biosynsis delivers fully validated and optimized enzyme cascade systems for industrial use. Every project includes:

• Cascade Design Report: Documentation of enzyme sources, reaction steps, and the rationale for co-factor regeneration and immobilization strategies.
• Optimized System: Delivery of plasmids or engineered microbial strains, or the co-immobilized enzyme preparation.
• Process Metrics: Detailed kinetics, reaction conditions, and quantitative data on final product yield, purity, and intermediate accumulation.
• Stability Data: Confirmation of the operational stability and reuse potential (if immobilized) of the final cascade system.