L-isoleucine in microbial fermentation production

L-isoleucine, an essential amino acid, is widely used in food, pharmaceutical, and feed industries. In microbial fermentation for L-isoleucine production, metabolic engineering strategies can optimize metabolic pathways and enhance yields. The following is a detailed introduction:

I. Enhancement of Precursor Synthesis

1. Strengthening Glycolysis

Glucose serves as the primary carbon source in microbial fermentation. Enhancing the expression of key enzymes in the glycolytic pathway can improve glucose metabolism efficiency, providing more precursors for L-isoleucine synthesis. For example, overexpressing the pyruvate kinase gene accelerates the breakdown of glucose into pyruvate, ensuring an abundant substrate supply for subsequent reactions.

2. Increasing Chorismate Supply

Chorismate is a critical precursor for L-isoleucine synthesis. Its production can be enhanced by strengthening the expression of key enzymes in the shikimate pathway. For instance, overexpressing the 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase gene promotes chorismate synthesis.

II. Regulation of Key Enzyme Genes

1. Overexpression of Rate-Limiting Enzyme Genes

In the L-isoleucine synthesis pathway, low activity of certain enzymes can become rate-limiting steps. Overexpressing these rate-limiting enzyme genes accelerates the reaction rate. Threonine dehydratase, for example, is a rate-limiting enzyme in L-isoleucine synthesis, and its overexpression can enhance the synthesis rate of L-isoleucine.

2. Regulation of Branch Metabolic Pathway Enzymes

Microbial systems often have branch metabolic pathways that compete with L-isoleucine for precursors. Inhibiting the activity or reducing the gene expression of key enzymes in these pathways redirects more precursors toward L-isoleucine synthesis. For example, gene knockout or RNA interference can suppress the expression of the threonine dehydrogenase gene, reducing threonine synthesis and shunting more precursors into L-isoleucine production.

III. Removing the Feedback Adjustment Mechanism (Removing the Feedback Adjustment Mechanism)

1. Construction of Feedback-Resistant Mutant Strains

Key enzymes in some amino acid synthesis pathways are subject to feedback inhibition by end products. Constructing feedback-resistant mutant strains through mutagenesis breeding or gene editing technologies can relieve this inhibition, enhancing enzyme activity and L-isoleucine yields. For example, engineering feedback-resistant mutations in aspartate kinase can eliminate its inhibition by L-isoleucine, boosting synthesis capacity.

2. Engineering of Regulatory Factors

Regulatory factors within microbial cells control the expression of metabolic pathway genes. Modifying these factors can alter their regulatory effects on L-isoleucine synthesis. Site-directed mutagenesis or modification of regulatory factors like Lrp (leucine-responsive regulatory protein) can relieve their inhibitory effects on genes involved in L-isoleucine synthesis.

IV. Optimization of Cofactors

1. Coenzyme Supplementation

Many enzymes involved in L-isoleucine synthesis require coenzymes, such as pyridoxal phosphate for transaminases. Adding appropriate coenzymes or their precursor substances to the fermentation medium can enhance enzyme activity and promote L-isoleucine synthesis.

2. Enhancing Coenzyme Regeneration Systems

Building efficient coenzyme regeneration systems ensures continuous coenzyme availability in cells. Introducing genes related to coenzyme regeneration, for example, maintains high coenzyme activity through continuous regeneration, improving the efficiency of L-isoleucine synthesis.

V. Fermentation Condition Optimization and Metabolic Flux Guidance

1. Fermentation Condition Optimization

Optimizing parameters such as temperature, pH, and dissolved oxygen during fermentation creates a suitable environment for microbial growth and L-isoleucine synthesis. Different microorganisms have varying requirements for fermentation conditions, and determining the optimal parameter combination through experiments can improve L-isoleucine yield and quality.

2. Metabolic Flux Guidance

Using metabolic flux analysis (MFA) to study intracellular metabolic flux distribution, researchers can adjust the activity of key enzymes or metabolite concentrations at metabolic nodes to redirect flux toward L-isoleucine synthesis. For example, controlling the ratio of carbon to nitrogen sources in the medium influences cellular metabolism, channeling more flux into L-isoleucine production.