Coumarin Bio-Production via Heterologous Expression Service

Coumarin is a widely used aromatic compound essential in the fragrance, food, and pharmaceutical industries. Traditional supply methods face significant drawbacks: low efficiency of extraction from plants , which relies on agricultural output, and many steps in chemical synthesis , leading to high costs and environmental waste. Securing a sustainable and efficient supply remains a challenge.

CD Biosynsis offers a robust synthetic biology solution to establish a reliable microbial production platform for coumarin. Our strategy is centered on the heterologous expression of coumarin synthase in Escherichia coli , creating a stable biological chassis for production. This is paired with the optimization of the shikimate pathway —the precursor supply route—to redirect carbon flux toward the desired intermediate. This integrated approach aims to establish a cleaner, more economical route for coumarin manufacturing.

Pain Points

Industrial supply of coumarin is limited by the inherent drawbacks of existing production methods:

• Unsustainable Plant Extraction: Extracting coumarin from natural sources (e.g., tonka beans, sweet clover) is highly dependent on variable agricultural yields and suffers from low purity and recovery rates.
• Complex Chemical Synthesis: The reliance on multi-step chemical routes (e.g., Perkin reaction) leads to high usage of organic solvents , complex waste streams, and high manufacturing costs.
• Precursor Bottleneck: The shikimate pathway, which supplies the aromatic precursor (e.g., p-coumaric acid) required for coumarin synthesis, is often tightly regulated in microbial hosts , limiting flux.
• Heterologous Gene Expression Issues: Successfully expressing complex plant-derived coumarin synthase enzymes in a microbial host like E. coli often results in low activity or poor protein folding.

A successful approach requires boosting the precursor supply and achieving highly active heterologous enzyme expression.

Solutions

CD Biosynsis employs synthetic biology tools to establish a functional and efficient coumarin pathway in E. coli:

Heterologous Expression of Coumarin Synthase

           

We clone, codon-optimize, and express the rate-limiting plant-derived coumarin synthase genes in E. coli, using strong promoters to maximize protein availability.

Optimization of the Shikimate Pathway

We use gene editing to downregulate competing pathways and upregulate key enzymes in the shikimate pathway, aiming to enhance the metabolic flux toward the coumarin precursor (p-coumaric acid).

Metabolic Channeling and Compartmentalization

We explore strategies to co-localize pathway enzymes using synthetic scaffolds or microcompartments, which may improve intermediate transfer and pathway efficiency.

Strain Tolerance and Product Removal

We investigate methods to increase E. coli's tolerance to coumarin and explore in situ product removal techniques to mitigate toxicity and facilitate recovery.

This systematic approach is focused on overcoming precursor supply and enzyme activity hurdles for coumarin bioproduction.

Advantages

Our coumarin engineering service is dedicated to exploring the following production benefits:

Sustainable Production Route

Offers a bio-based alternative that may potentially reduce the reliance on polluting chemical synthesis and volatile plant extraction.

Enhanced Precursor Supply

Shikimate pathway optimization aims to increase the availability of the crucial aromatic precursor needed for downstream synthesis.

Controlled Biosynthesis

Fermentation allows for precise, scalable control over reaction conditions, offering a level of consistency unattainable in plant extraction.

Targeted Metabolic Flux

Pathway engineering aims to successfully redirect central carbon flow to the high-value aromatic product.

Simplified Purification Potential

Using a clean microbial host may potentially reduce the complex mixture of contaminants found in plant extracts.

We provide a biosynthetic platform aimed at overcoming the limitations of current coumarin supply methods.

Process

Our Coumarin strain engineering service follows a standardized, multi-stage research workflow:

• Precursor Pathway Analysis: Conduct Flux Balance Analysis (FBA) to identify bottlenecks in the native shikimate pathway and design strategies for upregulating key steps.
• Coumarin Synthase Cloning and Expression: Screen and optimize the expression of multiple coumarin synthase variants from various plant sources to find the most active one in E. coli.
• Shikimate Pathway Engineering: Employ gene editing to de-regulate aromatic amino acid synthesis and enhance flux towards the precursor.
• Gene Circuit and Pathway Balancing: Implement genetic circuits to tune the expression of pathway enzymes to prevent intermediate accumulation and toxicity.
• Fermentation Feasibility Assessment: Test the final engineered strain in fed-batch fermentation to assess final titer, yield, and purity under controlled conditions.
• Result Report Output: Compile a detailed Experimental Report including genetic maps, enzyme activity data, fermentation kinetics, and a preliminary purification assessment , supporting further development.

Technical communication is maintained throughout the process, focusing on timely feedback regarding precursor supply and final product formation.

Explore the potential for sustainable, high-quality coumarin production. CD Biosynsis provides customized strain engineering solutions:

• Detailed Shikimate Pathway Flux Analysis Report , illustrating the successful redirection of central carbon resources.
• Consultation on fermentation strategies designed to manage any potential product toxicity.
• Experimental reports include complete raw data on titer, purity, and pathway enzyme activity , essential for assessing commercial viability.