Phenylalanine Bioproduction Engineering Service

Phenylalanine (Phe) is an essential aromatic amino acid (AAA) widely used in the pharmaceutical industry (e.g., precursors to Aspartame) and as a nutritional supplement. Industrial production via microbial fermentation, commonly using Escherichia coli (E. coli), is often hampered by a low acid production rate in fermentation . This is primarily due to the stringent feedback inhibition of Phe on its own biosynthetic enzymes. Furthermore, alternative methods like chemical synthesis are highly criticized for the high pollution generated from toxic solvents, heavy metal catalysts, and complex purification steps, making biosynthesis the preferred sustainable route.

CD Biosynsis offers a synthetic biology service focused on engineering E. coli for high-titer L-Phenylalanine production. Our core strategy involves modification of phenylalanine synthase in Escherichia coli . The key enzyme is Chorismate Mutase (CM)/Prephenate Dehydratase (PD) , which is part of the PheA enzyme complex (often referred to as Phenylalanine Synthase). CM is the rate-limiting step and is strongly inhibited by Phe. We use site-directed mutagenesis to create a feedback-resistant PheA variant (PheA ^mut) , ensuring the conversion remains active at high product concentrations. This is coupled with the optimization of shikimate pathway . Phe synthesis starts with the Shikimate Pathway (SP) generating Chorismate. We enhance the flux into SP by overexpressing DAHP Synthase (AroG or AroF variants) , which is the first committed and highly regulated enzyme in the SP. We also block competing pathways (e.g., Tryptophan and Tyrosine branches) to ensure maximum carbon flow is routed to Phe. This integrated strategy removes the metabolic brake (inhibition) and maximizes precursor availability, solving the low production rate problem and providing a sustainable alternative to high pollution chemical synthesis .

Pain Points

Achieving cost-effective, sustainable Phenylalanine production faces these key challenges:

• Low Acid Production Rate in Fermentation: Phenylalanine synthesis is tightly controlled by feedback inhibition on the PheA enzyme complex (CM/PD) and the first committed step of the SP (DAHP Synthase), leading to low titers.
• High Pollution in Chemical Synthesis: Chemical Phe production requires toxic reagents, harsh solvents, and complex downstream waste management , posing severe environmental risks.
• Precursor Supply Bottleneck: The synthesis of Chorismate from the Shikimate Pathway involves multiple steps, and achieving high flux requires overcoming multiple regulatory bottlenecks early in the pathway.
• Competition with other AAAs: Chorismate is a shared intermediate for Phe, Tyrosine, and Tryptophan synthesis, leading to carbon flux diversion to unwanted products.

A successful solution must neutralize key enzymatic inhibition sites and boost precursor supply from the SP.

Solutions

CD Biosynsis utilizes advanced metabolic engineering to optimize Phenylalanine production in E. coli:

Modification of Phenylalanine Synthase in E. coli

           

We mutate the PheA complex (CM/PD) to create a feedback-resistant PheA ^mut variant , allowing production to continue at high Phe concentrations.

Optimization of Shikimate Pathway

We overexpress feedback-resistant DAHP Synthase (AroG ^mut or AroF ^mut) to maximize carbon flux into the SP and Chorismate pool.

Competing AAA Pathway Blockade

We delete TyrA and TrpE to block the synthesis of Tyrosine and Tryptophan, directing all Chorismate toward Phenylalanine.

Efflux Pump Overexpression

We overexpress an AAA efflux system (e.g., AroP variants) to enhance Phe secretion, reducing internal toxicity and aiding yield.

This systematic approach overcomes multiple regulatory bottlenecks, maximizes precursor availability, and provides a sustainable production route.

Advantages

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

High Fermentation Titer

Removing feedback inhibition on both PheA and AroG ensures a fast, continuous acid production rate and high final yield.

Reduced Environmental Pollution

Biosynthesis entirely avoids the use of toxic chemicals and heavy metals from conventional chemical synthesis, offering a green, low pollution alternative.

High L-Phenylalanine Purity Icon

The engineered microbial pathway produces only the biologically active L-isomer , eliminating the need for complex racemic separation.

Economically Competitive Yield Icon

Optimized flux and high productivity lower manufacturing costs, making the biosynthetic product competitive with existing methods .

E. coli Platform Robustness Icon

Uses a well-characterized, robust, and scalable E. coli host for industrial fermentation.

We provide a sustainable, high-yield, and cost-effective manufacturing platform for L-Phenylalanine.

Process

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

• PheA Mutagenesis: Introduce site-directed mutations into the PheA enzyme (CM/PD) to eliminate feedback inhibition by Phe.
• Shikimate Pathway Optimization: Overexpress feedback-resistant AroG or AroF to maximize the flux of Erythrose-4-Phosphate (E4P) and Phosphoenolpyruvate (PEP) into the SP.
• Competing AAA Blockade: Delete TyrA and TrpE to completely block the synthesis of Tyrosine and Tryptophan from Chorismate.
• Precursor Supply and Efflux: Optimize the supply of E4P and PEP from central metabolism and overexpress Phe efflux pumps .
• Functional and Titer Assays: Validate the engineered strain in fed-batch culture, measuring the final L-Phenylalanine concentration and specific productivity .
• Result Report Output: Compile a detailed Experimental Report including gene modification data, enzyme characterization, and fermentation metrics (final titer, yield, and fermentation purity) , supporting industrial scale-up.

Technical communication is maintained throughout the process, focusing on timely feedback regarding yield and pathway efficiency.

Explore the potential for a cost-competitive, sustainable L-Phenylalanine supply. CD Biosynsis provides customized strain and pathway engineering solutions:

• Detailed Feedback Resistance and Flux Enhancement Report , demonstrating the functional change in PheA ^mut and the improved SP flux.
• Consultation on optimized carbon source and nutrient feeding strategies for maximizing E4P and PEP availability.
• Experimental reports include complete raw data on Phenylalanine productivity (g/L/h) and final product yield (g/g glucose) , highlighting the economic advantage over chemical synthesis.