Synthetic biology techniques of L-isoleucine

The breakthrough directions of synthetic biology technology for L-isoleucine mainly include the following aspects:

I. Modification of Key Enzymes

1. Relieving Feedback Inhibition

Key enzymes in the biosynthetic pathway of L-isoleucine, such as aspartate kinase (AK), homoserine dehydrogenase (HD), threonine dehydratase (TD), and acetohydroxyacid synthase (AHAS), are often subject to feedback inhibition by products like lysine, threonine, and L-isoleucine. Through genetic engineering technology, site-directed mutagenesis is performed on the coding genes of these key enzymes to alter the enzyme structure, reducing their sensitivity to feedback inhibitors. This relieves or weakens feedback inhibition, increases carbon flux, and enhances the accumulation of L-isoleucine.

2. Improving Enzyme Activity and Stability

Protein engineering technology is utilized to modify key enzymes, enhancing their catalytic activity and stability. For example, through directed evolution, rational design, and other methods, enzyme mutants with higher specific activity, more suitable reaction conditions (such as pH, temperature), and better stability are screened to accelerate the synthesis reaction of L-isoleucine and reduce the generation of by-products.

II. Optimization of Precursor Supply

1. Balancing Precursor Proportions

The synthesis of L-isoleucine requires various precursor substances, such as oxaloacetic acid and aspartic acid. Studies have found that adjusting the proportion of precursor substances is crucial for improving L-isoleucine yield. For instance, by screening key enzymes with high affinity for specific precursors, and knocking out or overexpressing related genes to regulate key enzymes in the precursor synthesis pathway, the precursor synthesis module is optimized to achieve balanced precursor supply, thereby increasing the yield and conversion rate of L-isoleucine.

2. Enhancing Precursor Synthesis Pathways

The synthesis pathways of precursor substances are strengthened to increase precursor supply. This can be achieved by overexpressing key enzyme genes in the precursor synthesis pathway to enhance the metabolic flux of precursor synthesis; or by introducing exogenous precursor synthesis pathways to bypass the original metabolic regulation nodes in cells and directly synthesize precursor substances efficiently, providing sufficient raw materials for L-isoleucine synthesis.

III. Regulation of Cellular Metabolic Flux

1. Reconstructing Metabolic Networks

Systematic analysis and reconstruction of the overall metabolic network of cells are carried out to break the original metabolic balance, guiding metabolic flux to flow more toward the L-isoleucine synthesis pathway. For example, by knocking out genes of metabolic pathways unrelated to L-isoleucine synthesis or competing for nutrients such as carbon and nitrogen sources, the generation of by-products is reduced, and more metabolic resources are allocated to L-isoleucine synthesis.

2. Dynamically Regulating Metabolic Flux

Dynamic regulatory elements in synthetic biology, such as promoters, riboswitches, and quorum sensing systems, are used to regulate the expression of key genes in the L-isoleucine synthesis pathway in real time and dynamically according to cell growth status and environmental conditions, achieving a balance between cell growth and product synthesis. For instance, in the early stage of cell growth, the L-isoleucine synthesis pathway is weakened to allow cells to preferentially use nutrients for growth and reproduction; when the cell density reaches a certain threshold, the L-isoleucine synthesis pathway is activated to synthesize the target product in large quantities.

IV. Strain Breeding and Modification

1. Screening Excellent Chassis Strains

Selecting appropriate microbial strains as chassis cells is the foundation of L-isoleucine synthetic biology. In addition to the commonly used Corynebacterium glutamicum and E. coli, strains with unique metabolic characteristics or potential advantages for L-isoleucine synthesis can also be screened from nature. Meanwhile, systematic physiological and biochemical characterization and genetic modification of existing production strains are conducted to improve their adaptability and tolerance to L-isoleucine synthesis.

2. Constructing Genetically Engineered Strains

Gene editing technologies such as CRISPR-Cas9 and homologous recombination are used to perform precise genetic operations on chassis strains, achieving coordinated expression, knockout, or replacement of multiple genes to construct genetically engineered strains for efficient L-isoleucine synthesis. Furthermore, by introducing exogenous genes or gene clusters, the metabolic pathways of strains can be expanded, endowing them with new metabolic functions and improving the synthesis efficiency of L-isoleucine.

V. Optimization of Fermentation Processes

1. Medium Optimization

In-depth research on the nutritional requirements of strains during L-isoleucine synthesis is conducted to optimize the composition of the medium, including the types and concentrations of carbon sources, nitrogen sources, inorganic salts, vitamins, and other components. A suitable ratio of carbon source to nitrogen source is selected to meet the needs of strain growth and L-isoleucine synthesis. Meanwhile, appropriate amounts of precursor substances, inducers, or other promoters are added to promote the synthesis and secretion of L-isoleucine.

2. Optimization of Fermentation Conditions

Various conditions in the fermentation process, such as temperature, pH, dissolved oxygen, and stirring speed, are finely regulated to create the most suitable environmental conditions for strain growth and L-isoleucine synthesis. For example, by controlling the fermentation temperature within the suitable range for strain growth and product synthesis, maintaining an appropriate pH value to ensure the activity of key enzymes, and optimizing the dissolved oxygen level to meet the needs of cell respiration and metabolic reactions, the yield and quality of L-isoleucine are improved.