Alanine Bioproduction Engineering Service

Alanine is a non-essential amino acid widely used in the food industry as a flavoring agent and in pharmaceuticals (e.g., infusion solutions). While L-Alanine can be produced via microbial fermentation, commercial viability is challenged by the low acid production rate in fermentation , leading to extended cycle times and high operational costs. Furthermore, conventional downstream processing is hindered by high extraction cost , particularly the need for complex separation processes to isolate the pure amino acid from the complex fermentation broth. Biosynthesis optimization offers a cleaner, higher-yield route.

CD Biosynsis offers a synthetic biology service focused on highly efficient Alanine production in Corynebacterium glutamicum, a powerful host for amino acid biosynthesis. Our core strategy involves metabolic regulation modification in Corynebacterium glutamicum . We genetically engineer the host to maximize the carbon flux from glucose toward pyruvate, a direct precursor for Alanine. This is achieved by deleting competing pathways (e.g., lactate or succinate production) and reinforcing rate-limiting steps in glycolysis. Crucially, this is coupled with overexpression of alanine dehydrogenase . We introduce and heavily overexpress a highly active Alanine Dehydrogenase (AlaDH) enzyme. AlaDH efficiently converts pyruvate directly into L-Alanine, the desired product, using NADH or NADPH as a cofactor, ensuring rapid, high-titer conversion and maximizing the final product concentration. This integrated approach aims to deliver high-titer, high-purity Alanine, significantly reducing both fermentation time and downstream purification expenses.

Pain Points

Developing a cost-effective Alanine production route faces these key limitations:

• Low Acid Production Rate in Fermentation: Alanine biosynthesis competes with central metabolic pathways, leading to slow conversion of substrate to product and low space-time yield in the fermenter.
• High Extraction Cost: Isolating pure Alanine from the complex, low-concentration fermentation broth requires multiple steps (e.g., filtration, ion-exchange chromatography, crystallization), resulting in high material and energy consumption .
• Pathway Competition: Pyruvate, the Alanine precursor, is rapidly consumed by competing pathways such as the TCA cycle, acetate, lactate, and succinate synthesis, lowering the overall carbon yield .
• Cofactor Imbalance: Alanine Dehydrogenase requires NADH or NADPH. Ensuring sufficient cofactor regeneration and availability is often a bottleneck for maximizing enzyme activity.

A successful solution must force the metabolic flux toward pyruvate and maximize the efficiency of the final enzymatic conversion step.

Solutions

CD Biosynsis utilizes advanced metabolic and enzyme engineering to optimize Alanine production in C. glutamicum:

Metabolic Regulation Modification in C. glutamicum

           

We delete competing pathways (ldh, pox, ppc) and overexpress key glycolytic enzymes (e.g., pyk) to channel maximum carbon flux from glucose directly to pyruvate.

Overexpression of Alanine Dehydrogenase

We introduce and overexpress a highly active, heterologous Alanine Dehydrogenase (AlaDH) under a strong promoter to ensure the rapid and efficient conversion of Pyruvate to L-Alanine.

Cofactor Engineering for NADH/NADPH Balance

We engineer the host's redox systems (e.g., transhydrogenase) to ensure optimal regeneration of the NADH/NADPH cofactor required by the overexpressed AlaDH.

Feed and Process Optimization

We develop optimized fed-batch strategies (e.g., continuous NH3 addition) to maintain the optimal pH and substrate concentration, minimizing product inhibition and maximizing titer.

This systematic approach is focused on rebuilding the central metabolism of C. glutamicum to function as a highly efficient L-Alanine production machine.

Advantages

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

High Fermentation Titer and Yield

Rerouting carbon flux and enzyme overexpression leads to a faster production rate and higher final concentration , solving the low yield issue.

Reduced Downstream Costs

Higher initial titer simplifies purification processes and lowers the total extraction cost , addressing the second major pain point. [Image of Cost Reduction Icon]

High Optical Purity

Enzymatic synthesis using AlaDH naturally yields high purity L-Alanine , avoiding the need for chiral resolution required in chemical synthesis.

Established Industrial Host

C. glutamicum is a non-pathogenic, generally recognized as safe (GRAS) organism with decades of use in large-scale amino acid production.

Sustainable Production

Uses renewable carbon sources (e.g., glucose) as feedstocks, promoting an environmentally friendly biomanufacturing process.

We provide a sustainable and cost-effective biosynthetic platform for industrial L-Alanine production.

Process

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

• Metabolic Pathway Knockouts: Use CRISPR-Cas systems to delete genes for competing pathways (e.g., lactate dehydrogenase) and optimize carbon flow to pyruvate.
• Gene Overexpression: Introduce and overexpress the heterologous Alanine Dehydrogenase (AlaDH) gene under strong promoters to maximize the final conversion rate.
• Cofactor Balance Tuning: Engineer specific enzymes to enhance NADH regeneration within the cell, meeting the high demands of the AlaDH reaction.
• Process Optimization: Develop and test fed-batch strategies, focusing on substrate concentration, pH control, and aeration rate to maximize AlaDH activity and product accumulation.
• Fermentation Performance Validation: Test the final engineered strain in bench-scale bioreactors to assess Alanine titer, yield, and optical purity .
• Result Report Output: Compile a detailed Experimental Report including gene modification data, metabolic flux analysis, and fermentation metrics (yield, titer, and productivity) , supporting commercial scale-up.

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

Explore the potential for a high-titer, high-purity Alanine supply. CD Biosynsis provides customized strain and process engineering solutions:

• Detailed Titer and Yield Analysis Report , demonstrating the success of metabolic flux rerouting toward L-Alanine.
• Consultation on optimized cell recovery and purification protocols to lower downstream costs.
• Experimental reports include complete raw data on carbon yield (g Alanine/g glucose) and optical purity (ee} \%$) , essential for pharmaceutical grade products.