Isoleucine Bioproduction Engineering Service

Isoleucine (Ile) is an essential branched-chain amino acid (BCAA) widely used in animal feed and human health products. Industrial microbial fermentation, typically utilizing Corynebacterium glutamicum, is often hampered by a low acid production rate in fermentation . This is due to complex allosteric regulation, where Isoleucine inhibits its own synthesis pathway, primarily at the enzyme Threonine Deaminase (IlvA). Furthermore, the structural similarity to other BCAAs (Valine, Leucine) and their co-production can lead to difficult extraction and purification , driving up manufacturing costs. A precise metabolic engineering approach is necessary to overcome these yield and purity hurdles.

CD Biosynsis offers a synthetic biology service focused on high-titer L-Isoleucine production in C. glutamicum. Our core strategy involves metabolic flow regulation of lysine synthesis enzyme in Corynebacterium glutamicum . This strategy is multi-faceted. First, we target the main bottleneck: we use site-directed mutagenesis on Threonine Deaminase (IlvA) to make it feedback-resistant to Isoleucine inhibition, drastically increasing the Ile flux. Second, we focus on the shared precursor pool. Since Isoleucine synthesis starts with Threonine (which shares Aspartate as a precursor with Lysine and Methionine), we employ metabolic flow regulation of the Lysine synthesis enzyme (e.g., Aspartate Kinase, LysC) to ensure maximum carbon flux is diverted towards Threonine and subsequently to Isoleucine. This involves deleting or downregulating Lysine and Methionine synthesis genes and upregulating Threonine production. Finally, we implement efflux pump overexpression to improve product secretion, easing the downstream burden of difficult extraction and purification. This comprehensive regulation strategy significantly boosts both the fermentation titer and the purity of the final L-Isoleucine product.

Pain Points

Achieving cost-effective, high-yield Isoleucine production faces these key challenges:

• Low Acid Production Rate in Fermentation: The key enzyme, Threonine Deaminase (IlvA) , is highly sensitive to feedback inhibition by Isoleucine , leading to low volumetric productivity.
• Difficult Extraction and Purification: Co-production of Valine and Leucine and the similar physiochemical properties of BCAAs make their separation complex, energy-intensive, and costly .
• Precursor Competition: Isoleucine synthesis relies on Threonine as a precursor, which is in competition with the Lysine and Methionine synthesis pathways for the common Aspartate intermediate.
• Pathway Blockage: Lack of efficient product export leads to intracellular accumulation and product toxicity , further slowing cell growth and production rate.

A successful solution must remove feedback control and ensure maximal flux from the Aspartate pathway is directed to Isoleucine.

Solutions

CD Biosynsis utilizes advanced metabolic engineering to optimize Isoleucine production in C. glutamicum:

IlvA Feedback Resistance Modification

           

We mutate IlvA (Threonine Deaminase) to abolish Isoleucine feedback inhibition, thus releasing the main metabolic bottleneck.

Metabolic Flow Regulation of Lysine Synthesis Enzyme

We delete or downregulate LysC (Aspartate Kinase) and Methionine pathway genes (e.g., MetB) to divert Aspartate flux toward Threonine (Isoleucine precursor).

Competing BCAA Pathway Blockade

We delete or downregulate the Valine and Leucine synthesis branches to ensure the a-ketobutyrate and a-ketoisovalerate intermediates are channeled to Isoleucine.

Enhanced Product Efflux

We overexpress BCAA efflux pumps to promote Isoleucine secretion, reducing internal toxicity and simplifying downstream purification.

This systematic approach optimizes the entire biosynthetic network from the primary precursor to the final product secretion.

Advantages

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

High Fermentation Titer

Removal of IlvA feedback inhibition allows for a continuous, high acid production rate , solving the low-yield issue.

Simplified Purification

Strict Metabolic Flow Regulation reduces Valine and Leucine side-products, making extraction and purification much easier and cheaper. [Image of Cost Reduction Icon]

Purity and L-Isomer Guarantee Icon

Biosynthesis yields only the biologically active L-Isoleucine , free from inactive enantiomers and reduced co-produced BCAAs purity.

Efficient Precursor Utilization Icon

Regulating the Aspartate pathway ensures the feedstock is channeled efficiently towards the Isoleucine branch.

Reduced Product Toxicity

Active product efflux helps prevent intracellular buildup, maintaining cell health and productivity.

We provide a sustainable, high-purity, and cost-efficient L-Isoleucine manufacturing solution.

Process

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

• IlvA Mutagenesis: Introduce site-directed mutations into Threonine Deaminase (IlvA) to render it feedback-resistant .
• Aspartate Pathway Regulation: Delete/downregulate LysC and MetB to maximize Aspartate flux into Threonine synthesis.
• Competing BCAA Blockade: Knock out key enzymes in the Valine and Leucine synthesis branches to ensure specificity to Isoleucine.
• Pathway Overexpression: Amplify the expression of Isoleucine specific enzymes (IlvA^mut, IlvB, IlvC, IlvD, IlvE) to increase pathway throughput.
• Efflux Pump Integration: Overexpress a BCAA efflux pump to enhance Isoleucine secretion and reduce internal toxicity.
• Functional and Titer Assays: Validate the engineered strain in fed-batch culture, measuring the final L-Isoleucine concentration and purity (low Valine/Leucine content) .
• Result Report Output: Compile a detailed Experimental Report including gene modification data, enzyme characterization, and fermentation metrics (final titer, yield, and co-product ratio) , 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 high-titer, high-purity L-Isoleucine supply. CD Biosynsis provides customized strain and pathway engineering solutions:

• Detailed Feedback Resistance and Flux Partitioning Report , demonstrating the functional change in IlvA^mut and the efficiency of Aspartate partitioning.
• Consultation on optimized amino acid supplementation strategies to minimize carbon diversion to unwanted pathways.
• Experimental reports include complete raw data on Isoleucine productivity (g/L/h) and BCAA co-product profile , essential for purity validation and downstream cost estimation.