L-isoleucine in microbial fuel cells

The metabolic regulation of L-isoleucine in microbial fuel cells (MFCs) involves multiple aspects, which are specifically introduced as follows:

I. Regulation of Microbial Metabolic Pathways

1. Enhancing Central Carbon Metabolic Flux

The synthetic precursors of L-isoleucine are closely associated with the central carbon metabolic pathways of microorganisms. For example, in some microbes, strengthening the glycolytic pathway and tricarboxylic acid cycle (TCA) can direct more carbon sources toward L-isoleucine synthesis. Overexpressing key enzyme genes related to this process promotes the efficient conversion of carbon sources like glucose into pyruvate, providing abundant precursors for L-isoleucine synthesis. Meanwhile, this supplies more electron donors for MFCs, enhancing battery performance.

2. Optimizing Precursor Supply

L-isoleucine synthesis requires various precursors, such as aspartic acid and pyruvate. Genetic engineering can be used to modify or regulate key enzymes in precursor synthesis pathways, improving precursor synthesis efficiency and supply. For instance, enhancing the expression of aspartate kinase genes increases aspartic acid production, providing more substrates for L-isoleucine synthesis. This helps improve its yield in MFCs and enhance battery performance.

II. Improving Activity and Expression of Key Enzymes

1. Relieving Feedback Inhibition of Key Enzymes

Key enzymes in the L-isoleucine synthesis pathway, such as acetohydroxyacid synthase (AHAS), are often inhibited by feedback from products like L-isoleucine. Site-directed mutagenesis and other techniques can modify the coding genes of these enzymes, altering their structure to reduce sensitivity to feedback inhibitors. This relieves or weakens feedback inhibition, increasing L-isoleucine synthesis flux and accumulation. It may also affect electron transfer and energy conversion efficiency in related metabolic processes of MFCs.

2. Enhancing Key Enzyme Expression

Genetic engineering techniques, such as placing key enzyme genes of the L-isoleucine synthesis pathway under the control of strong promoters or increasing gene copy numbers, can improve key enzyme expression. This accelerates L-isoleucine synthesis, enabling microbes to produce it more efficiently and potentially enhancing MFC performance.

III. Modifying Cell Membrane Permeability

1. Regulating Transporters

After intracellular synthesis, L-isoleucine needs to be promptly transported outside the cell to avoid feedback inhibition from product accumulation. Strengthening the expression or activity of L-isoleucine transporters within the cell allows rapid export of L-isoleucine, relieving feedback inhibition and promoting continuous synthesis. This also helps maintain metabolic balance in microbial cells, ensuring stable MFC operation.

2. Controlling Cell Membrane Components

Limiting the biotin concentration in the fermentation medium or adding appropriate surfactants can affect phospholipid synthesis in the cell membrane, thereby altering membrane permeability. For example, restricting biotin concentration in the late logarithmic phase enhances bacterial membrane permeability, facilitating extracellular secretion of L-isoleucine and increasing its yield. This may influence the interaction between microbes and electrodes in MFCs, thereby affecting battery performance.

IV. Optimizing Fermentation Conditions

1. Controlling Dissolved Oxygen (DO)

L-isoleucine synthesis is closely related to microbial respiratory metabolism, and DO levels affect intracellular redox status and energy metabolism. In MFCs, DO concentration must be finely regulated according to the microbial strain and fermentation stage. For instance, higher DO concentrations can be maintained during the bacterial growth phase to meet energy demands, while moderate DO reduction during L-isoleucine synthesis may promote its production. This also impacts electron transfer and power generation efficiency in MFCs.

2. Adjusting pH

pH affects enzyme activity, cell membrane stability, and nutrient absorption/metabolite synthesis in microbial cells. Maintaining an appropriate pH range is crucial for both L-isoleucine synthesis and MFC performance. Typically, different pH values should be controlled at various fermentation stages based on microbial characteristics to optimize L-isoleucine synthesis and microbial metabolic activities, thereby enhancing MFC performance.