Muconic Acid Bioproduction Engineering Service

Muconic Acid (MA) is a crucial intermediate for producing Adipic Acid (a Nylon precursor) and terephthalic acid (PET precursor). Bio-based MA production via the Shikimate pathway is promising but faces challenges: Low yield from biological synthesis in E. coli due to flux bottlenecks in the native pathway. Additionally, MA is toxic to cells; product toxicity at high concentrations inhibits cell growth , and the polar nature of MA causes high cost of separation from fermentation broth .

CD Biosynsis offers a systematic metabolic engineering approach to optimize MA production. Our core strategy includes: Metabolic Engineering: Overexpress key enzymes in the Shikimate pathway (e.g., DAHP Synthase) to increase carbon flux to Chorismate . This maximizes the MA precursor supply. We then Introduce a three-step heterologous pathway (PheA, PheC, CatA) for conversion from Chorismate to Muconic Acid . This synthetic route efficiently directs the precursor to the final product. Crucially, we use Efflux System: Engineering Muconic Acid transporters for enhanced secretion and reduced toxicity . This protects the cell from MA accumulation, allowing for higher titer production and simplifying downstream purification.

Pain Points

Industrial Muconic Acid production via biosynthesis faces these key challenges:

• Flux Bottlenecks in Shikimate Pathway: The initial steps of the native Shikimate pathway (e.g., DAHP Synthase) are highly regulated and feedback-inhibited, resulting in low carbon flux to the Chorismate precursor .
• Product Toxicity: Muconic Acid (especially at low pH) is toxic to microbial hosts like E. coli, causing cell membrane damage and growth inhibition at high titers.
• High Separation Cost: MA (a dicarboxylic acid) is highly soluble in water. Extracting it from the aqueous fermentation broth is energy-intensive and dominates the overall production cost .
• Limited Conversion Efficiency: The heterologous enzymes (e.g., CatA or PheA) needed for conversion from Chorismate to MA may have suboptimal activity or stability in the host.

A successful solution requires overcoming metabolic bottlenecks, enhancing cellular tolerance, and simplifying downstream recovery.

Solutions

CD Biosynsis utilizes advanced metabolic and enzyme engineering to optimize Muconic Acid production:

Shikimate Pathway Flux Engineering

           

We overexpress and deregulate key enzymes (e.g., feedback-resistant DAHP Synthase and Chorismate Mutase) to maximize carbon flow from glucose to Chorismate .

Synthetic Conversion Pathway (Chorismate to MA)

We introduce a three-step heterologous pathway (PheA, PheC, CatA or equivalent enzymes) to channel Chorismate to cis,cis-Muconic Acid with high selectivity.

Efflux System and Tolerance Engineering

We engineer or overexpress native or heterologous membrane transporters to actively secrete Muconic Acid out of the cell, minimizing intracellular toxicity and enabling high titers.

Enzyme Engineering for Stability

We optimize codon usage and use directed evolution to enhance the activity and acid tolerance of key enzymes like CatA in the host (E. coli) environment. [Image of High Conversion Efficiency Icon]

This integrated approach addresses flux, toxicity, and separation challenges for efficient MA production.

Advantages

Our Muconic Acid engineering service is dedicated to pursuing the following production goals:

Maximized Carbon Flux Icon

Deregulating Shikimate pathway enzymes guarantees a significantly higher precursor supply (Chorismate) to the MA pathway.

Increased Product Titer and Yield Icon

Optimized flux and reduced toxicity via efflux pumps enable cells to produce MA at higher concentrations.

Simplified Separation Process Icon

Active secretion (efflux) minimizes intracellular MA accumulation and makes downstream recovery more efficient. [Image of Cost Reduction Icon]

Bio-Based Monomer for Nylon/PET Icon

Provides a sustainable and environmentally friendly alternative to petrochemical Adipic Acid synthesis .

Optimized Enzyme Performance Icon

Enzyme engineering removes bottlenecks in the heterologous conversion of Chorismate to Muconic Acid.

We provide a competitive biological route for high-quality Muconic Acid production.

Process

Our Muconic Acid strain engineering service follows a rigorous, multi-stage research workflow:

• Precursor Flux Enhancement: Employ CRISPR/Cas or plasmid-based overexpression of a feedback-resistant DAHP Synthase (AroG fbr or AroF fbr) to maximize flux to Shikimate and Chorismate.
• Pathway Construction: Introduce and tune the expression levels of the three heterologous genes (PheA, PheC, CatA) on an optimized synthetic operon to ensure balanced conversion.
• Toxicity Mitigation: Screen and engineer Muconic Acid efflux transporters (e.g., MFS or ABC transporters) to actively pump MA out of the cell, reducing intracellular concentration.
• Enzyme Optimization: Use high-throughput methods (e.g., recombination and screening) to improve the thermostability and acid tolerance of key ring-cleaving enzyme (CatA) .
• Fermentation Validation: Validate the engineered strain in fed-batch fermentation to measure the final MA titer (g/L), yield, and productivity (g/L/h) under industrial conditions.

Technical communication is maintained throughout the process, focusing on timely feedback regarding yield and product quality attributes.

Explore the potential for a high-titer, bio-based Muconic Acid supply. CD Biosynsis provides customized strain and process engineering solutions:

• Detailed Enzyme Kinetic Report for the final optimized pathway enzymes, demonstrating enhanced activity (kcat and Km) and flux.
• Consultation on efflux pump expression and fermentation control strategies to manage Muconic Acid toxicity.
• Experimental reports include complete raw data on final MA titer, yield (g/g glucose), and productivity , crucial for economic assessment.