Microorganisms can rapidly release excess carbon and generate ATP through acetate-producing pathways, while also activating extracellular acetate into acetyl-CoA. Phosphotransacetylase, acetate kinase, and acetyl-CoA synthetase jointly regulate acetate production, reutilization, and the distribution of central carbon metabolic flux.

Keywords: acetate metabolism; phosphotransacetylase; acetate kinase; acetyl-CoA synthetase; acetyl phosphate; acetyl-CoA; substrate-level phosphorylation; overflow metabolism; microbial fermentation; metabolic engineering

1 Major Pathways and Physiological Significance of Microbial Acetate Metabolism

1.1 Acetate-Producing Pathways

Acetate in microorganisms is mainly derived from acetyl-CoA, acetyl phosphate, or acetaldehyde. Pyruvate generated by glycolysis can be converted into acetyl-CoA by pyruvate dehydrogenase. In some anaerobic microorganisms, pyruvate formate-lyase can also generate acetyl-CoA. Acetyl-CoA is subsequently converted into acetate through sequential reactions catalyzed by phosphotransacetylase and acetate kinase, accompanied by ATP generation. Some microorganisms can also convert pyruvate into acetyl phosphate through pyruvate oxidase, after which acetate kinase generates acetate. Acetic acid bacteria and some yeasts and bacteria can sequentially oxidize ethanol and acetaldehyde through alcohol dehydrogenase and aldehyde dehydrogenase, ultimately producing acetate.

1.2 Acetate-Utilizing Pathways

Extracellular acetate must first be converted into acetyl-CoA before entering the tricarboxylic acid cycle, glyoxylate cycle, fatty acid synthesis, and other acetyl-CoA-dependent pathways. Acetyl-CoA synthetase is the principal enzyme responsible for utilizing low concentrations of acetate and directly catalyzes the formation of acetyl-CoA from acetate, coenzyme A, and ATP. At relatively high acetate concentrations, some microorganisms can also use the reverse reactions of acetate kinase and phosphotransacetylase, converting acetate first into acetyl phosphate and then into acetyl-CoA.

Figure 1. Acetate activation and entry into the TCA cycle

1.3 Physiological Significance of Acetate Metabolism

(1) Energy Generation

When acetyl-CoA is converted into acetate through the phosphotransacetylase-acetate kinase pathway, ATP can be generated by substrate-level phosphorylation. This pathway plays an important energy-supplying role under anaerobic, oxygen-limited, or respiration-deficient conditions.

(2) Carbon Flux Distribution

When the rate of acetyl-CoA formation through glycolysis exceeds the processing capacity of the tricarboxylic acid cycle and respiratory chain, excess carbon can be excreted in the form of acetate. After depletion of the primary carbon source, some microorganisms can take up acetate again, making acetate a dynamic metabolic node connecting carbon overflow and carbon recovery.

(3) Coenzyme A Regeneration

When phosphotransacetylase converts acetyl-CoA into acetyl phosphate, free coenzyme A is released. This process helps maintain pyruvate oxidation, fatty acid degradation, and other coenzyme A-dependent reactions.

(4) Environmental Adaptation

Acetate accumulation can decrease the pH of the culture medium. Undissociated acetic acid can cross the cell membrane and dissociate intracellularly, increasing the energy required for proton export and maintenance of ionic homeostasis and thereby inhibiting microbial growth and target-product synthesis.

1.4 Pathway Differences Among Microorganisms

Most bacteria contain both the phosphotransacetylase-acetate kinase pathway and the acetyl-CoA synthetase pathway, but the expression levels and physiological functions of these pathways vary among species. Yeasts and filamentous fungi generally depend on acetyl-CoA synthetase for acetate assimilation and use aldehyde dehydrogenase for acetate formation. Some archaea and strictly anaerobic microorganisms contain ADP-forming acetyl-CoA synthetase, which directly catalyzes the formation of acetate, ATP, and coenzyme A from acetyl-CoA, ADP, and inorganic phosphate. This enzyme differs from the common AMP-forming acetyl-CoA synthetase in reaction direction and energy-coupling mechanism.

2 Classification and Reaction Characteristics of Enzymes Related to Acetate Production and Utilization

2.1 Enzymes Related to Acetate Production

Phosphotransacetylase catalyzes reversible acetyl-group transfer between acetyl-CoA and acetyl phosphate. Acetate kinase subsequently catalyzes the formation of acetate and ATP from acetyl phosphate and ADP. Together, these enzymes form an important bacterial pathway for acetate production and substrate-level phosphorylation. Aldehyde dehydrogenase oxidizes acetaldehyde into acetate while generating NADH or NADPH. Pyruvate oxidase converts pyruvate into acetyl phosphate, providing a direct substrate for acetate kinase.

2.2 Enzymes Related to Acetate Utilization

Acetyl-CoA synthetase catalyzes the formation of acetyl-CoA, AMP, and pyrophosphate from acetate, coenzyme A, and ATP. This reaction proceeds through an acetyl-adenylate intermediate and generally exhibits high affinity for acetate. In some bacteria, the acetate kinase-phosphotransacetylase pathway can also operate in reverse. This pathway generally has lower acetate affinity than acetyl-CoA synthetase but can provide a relatively high acetate-activation flux at elevated acetate concentrations.

2.3 Enzymes Related to Downstream Acetyl-CoA Utilization

When acetyl-CoA enters the tricarboxylic acid cycle, citrate synthase first condenses it with oxaloacetate to form citrate. When acetate is the principal carbon source, the glyoxylate cycle formed by isocitrate lyase and malate synthase reduces carbon loss as CO₂, allowing acetate-derived carbon to support gluconeogenesis and biomass synthesis. Acetyl-CoA can also enter pathways for fatty acid, polyhydroxyalkanoate, isoprenoid, and other secondary metabolite biosynthesis. Therefore, the efficiency of acetate activation directly affects the biosynthetic capacity for acetyl-CoA-derived products.

2.4 Functional Comparison of Key Enzymes

Table 1 Reaction Characteristics of Key Enzymes in Microbial Acetate Metabolism

3 Metabolic Functions of the Phosphotransacetylase-Acetate Kinase Pathway

3.1 Acetyl-Group Transfer Mediated by Phosphotransacetylase

Phosphotransacetylase (PTA) catalyzes the reversible reaction "acetyl-CoA + Pi ⇌ acetyl phosphate + CoA." It converts the high-energy thioester bond in acetyl-CoA into the high-energy acyl phosphate bond in acetyl phosphate while releasing free coenzyme A. The generated acetyl phosphate can serve as a substrate for acetate kinase and can also participate in two-component regulatory systems, protein acetylation, and other metabolic regulatory processes.

3.2 Substrate-Level Phosphorylation Mediated by Acetate Kinase

Acetate kinase (ACK) catalyzes the reversible reaction "acetyl phosphate + ADP ⇌ acetate + ATP." In the forward reaction, the phosphate group of acetyl phosphate is transferred to ADP, generating ATP and acetate. This reaction does not depend on the electron transport chain and can provide additional ATP during fermentative metabolism or when respiratory capacity is insufficient. In the reverse reaction, ATP is consumed to convert acetate into acetyl phosphate, which can then be converted into acetyl-CoA by PTA. However, this direction generally requires relatively high acetate concentrations.

3.3 Acetate Overflow Metabolism

When glucose uptake exceeds the processing capacity of the tricarboxylic acid cycle, respiratory chain, and biosynthetic pathways, excess acetyl-CoA enters the PTA-ACK pathway. This process rapidly regenerates coenzyme A and generates ATP but causes acetate excretion. Acetate overflow does not occur exclusively under oxygen-limited conditions. High glucose concentrations, limited respiratory capacity, altered protein-resource allocation, and changes in intracellular metabolite ratios can also promote acetate production.

3.4 Regulatory Functions of Acetyl Phosphate

Acetyl phosphate is not only an intermediate in acetate metabolism but can also donate phosphate groups to response regulators in certain two-component regulatory systems. Changes in acetyl phosphate levels may affect chemotaxis, motility, stress responses, biofilm formation, and expression of virulence-related genes. Acetyl phosphate can also participate in nonenzymatic lysine acetylation of proteins. Therefore, changes in PTA or ACK activity may further alter the acetylation states of metabolic enzymes and transcriptional regulators.

3.5 Pathway Direction and Metabolite Concentrations

The net direction of the PTA-ACK pathway is jointly influenced by the concentrations of acetyl-CoA, acetyl phosphate, acetate, ATP, ADP, coenzyme A, and inorganic phosphate. High-glucose and high-acetyl-CoA conditions generally promote acetate production, whereas reverse acetate utilization may increase when extracellular acetate concentrations are high and other carbon sources are limited. Measurement of pta or ackA gene expression alone cannot accurately determine pathway direction. Enzyme activity, extracellular acetate concentration, and intracellular metabolite changes should be analyzed together.

4 Mechanism of Acetate Activation Mediated by Acetyl-CoA Synthetase

4.1 Acetate Adenylation

Acetyl-CoA synthetase (ACS) first catalyzes the reaction "acetate + ATP → acetyl-AMP + PPi," forming an enzyme-bound acetyl-adenylate intermediate. Adenylation of the acetate carboxyl group increases the reactivity of the acetyl group and enables subsequent nucleophilic attack by coenzyme A and acetyl-group transfer.

4.2 Transfer of the Acetyl Group to Coenzyme A

Coenzyme A subsequently attacks the acetyl-adenylate intermediate, completing the reaction "acetyl-AMP + CoA → acetyl-CoA + AMP." The generated pyrophosphate can be further hydrolyzed into inorganic phosphate, driving the overall reaction toward acetyl-CoA formation. Because ATP is converted into AMP, this process consumes the equivalent of two high-energy phosphate bonds.

4.3 High-Affinity Acetate Uptake

ACS generally has a high affinity for acetate and is therefore suitable for uptake and removal of low concentrations of acetate. During batch culture, when glucose or another primary carbon source is depleted, some microorganisms increase ACS expression and reutilize acetate excreted during the early phase of cultivation. The generated acetyl-CoA can enter the tricarboxylic acid cycle and glyoxylate cycle to support continued cell growth. Therefore, a transition from acetate accumulation to acetate consumption generally indicates that the culture has entered a later stage of carbon-source switching.

4.4 Transcriptional Regulation of ACS

ACS expression is commonly regulated by carbon-source status, growth stage, and global carbon-metabolism regulatory networks. When readily utilized carbon sources are abundant, ACS expression may be repressed by carbon catabolite repression. After depletion of the primary carbon source or when acetate becomes the principal carbon source, ACS expression generally increases. Because transcription factors and promoter structures differ among microorganisms, ACS regulation should be evaluated in the context of the specific species, culture conditions, and growth stage.

4.5 Regulation of ACS by Reversible Acetylation

Some bacteria regulate ACS activity through reversible acetylation of a critical lysine residue. Acetylation catalyzed by acetyltransferases generally inhibits ACS, whereas NAD⁺-dependent deacetylases can restore catalytic activity. This post-translational regulation rapidly adjusts acetate-activation flux without changing protein abundance. Therefore, when measuring total ACS protein, enzyme activity or acetylation status should be evaluated simultaneously to avoid equating protein expression directly with actual catalytic capacity.

5 Energy Yields and Regulatory Differences Among Acetate Metabolic Pathways

5.1 Energy Yield of the Forward PTA-ACK Pathway

When acetyl-CoA is converted into acetate through the PTA-ACK pathway, one ATP molecule is generated per acetate molecule. This ATP is produced by substrate-level phosphorylation and does not directly depend on oxidative phosphorylation. The pathway rapidly recovers part of the available energy, but during acetate excretion, the carbon derived from acetyl-CoA cannot continue through the tricarboxylic acid cycle to generate additional reducing equivalents and ATP. If acetate is subsequently taken up and reactivated to acetyl-CoA, it can re-enter central carbon metabolism. Its advantage is a high reaction rate, whereas its cost is reduced carbon-use efficiency.

5.2 Energy Cost of the ACS Pathway

ACS converts ATP into AMP and pyrophosphate, corresponding to consumption of two high-energy phosphate-bond equivalents. Although this pathway has a relatively high energy cost, it efficiently captures low concentrations of acetate and is suitable for reutilizing extracellular acetate. Its actual flux is also limited by ATP availability, coenzyme A concentration, and the rate of downstream acetyl-CoA consumption.

5.3 Energy Cost of the Reverse PTA-ACK Pathway

During conversion of acetate into acetyl-CoA through the reverse PTA-ACK pathway, acetate kinase consumes ATP and generates ADP. Compared with the ACS pathway, this pathway consumes fewer high-energy phosphate bonds but generally has lower acetate affinity and is therefore more suitable at relatively high acetate concentrations. Under low-acetate conditions, ACS is usually the more effective acetate-uptake pathway.

5.4 Growth-Stage-Dependent Pathway Switching

During high-glucose growth, acetate production generally predominates, and PTA and ACK participate in excess-carbon excretion and ATP formation. As the primary carbon source is gradually depleted, acetate-production flux decreases, expression of ACS and glyoxylate-cycle enzymes increases, and cells begin consuming accumulated acetate. Time-dependent changes in acetate concentration are more informative than a single endpoint concentration. Continuous accumulation, a plateau phase, and a later decrease indicate sustained acetate production, a balance between production and utilization, and enhanced acetate reutilization, respectively.

5.5 Comparison of Energy and Regulatory Characteristics

Table 2 Functional Differences Among Acetate Metabolic Pathways

6 Applications of Key Acetate Metabolic Enzymes in Fermentation, Carbon Metabolism, and Metabolic Engineering

6.1 High-Cell-Density Culture and Recombinant Protein Production

In high-cell-density bacterial cultures, excessive carbon-source supply often causes acetate accumulation. Acetate can inhibit cell growth, reduce recombinant protein expression, and increase cellular maintenance-energy requirements. Therefore, controlling carbon-source uptake and PTA-ACK flux is an important aspect of fermentation optimization. Reducing the feeding rate, improving oxygen supply, enhancing respiratory metabolism, or using host strains that produce less acetate can decrease acetate accumulation. Direct deletion of pta or ackA may reduce acetate production but can also affect ATP supply, coenzyme A regeneration, and acetyl phosphate-mediated regulation.

6.2 Acetate and Organic Acid Fermentation

When acetate is the target product, carbon flux from acetyl-CoA toward acetate production can be enhanced while acetate reutilization is reduced, together with improvements in acid tolerance and acetate-export capacity. Acetic acid fermentation using ethanol as the substrate mainly depends on alcohol dehydrogenase and aldehyde dehydrogenase. This pathway differs from the PTA-ACK pathway in carbon source, electron transfer, and energy-generation mechanism, and regulatory strategies should therefore be designed according to the production organism.

6.3 Acetate as an Alternative Carbon Source

Acetate can be derived from anaerobic digestion, syngas fermentation, biomass pyrolysis liquids, and other industrial processes. Enhancing ACS, the glyoxylate cycle, and downstream acetyl-CoA-utilizing pathways can convert acetate into fatty acids, polyhydroxyalkanoates, terpenoids, and other bio-based products. Acetate assimilation consumes ATP and increases acid stress. Therefore, improving acetate uptake alone is insufficient; ATP supply, proton export, coenzyme A regeneration, and target-product synthesis flux must also be optimized.

6.4 Reduction of Acetate By-Product Formation

In the production of amino acids, nucleotides, enzyme preparations, and recombinant proteins, acetate is generally an undesirable by-product. Acetate formation can be reduced by decreasing glucose uptake, enhancing the tricarboxylic acid cycle, increasing respiratory capacity, or optimizing acetyl-CoA distribution. A decrease in acetate concentration does not necessarily indicate improved carbon utilization. If biomass and target-product formation decrease simultaneously, the modification may have merely reduced overall carbon metabolism. Carbon yield and product yield should therefore be considered together.

6.5 Improvement of Acetyl-CoA-Derived Product Synthesis

Enhanced ACS expression can directly introduce extracellular acetate into the acetyl-CoA metabolic pool. Use of highly active ACS variants or ACS variants resistant to inactivation by acetylation can improve the conversion of low concentrations of acetate. After acetate activation is enhanced, the target-product pathway must also be strengthened and futile cycling reduced. Otherwise, excess acetyl-CoA may re-enter acetate-producing pathways or increased ATP consumption may limit cell growth.

6.6 Stable-Isotope Metabolic Tracing

¹³C-labeled acetate can be used to trace acetate-derived carbon entering acetyl-CoA, the tricarboxylic acid cycle, lipids, and target products. Combined with mass spectrometric analysis of isotopic distributions, this approach can evaluate acetate uptake rates and the contributions of different acetate-activation pathways. Tracing experiments should consider dilution by unlabeled carbon sources, exchange among metabolic pools, and changes across culture stages. Extracellular acetate, intracellular acetyl-CoA, and the labeling proportions of downstream metabolites should be measured simultaneously.

7 Activity Assays for Acetate Metabolic Enzymes and Evaluation of Metabolic Products

7.1 Phosphotransacetylase Activity Assays

PTA activity can be evaluated by measuring acetyl-CoA consumption, coenzyme A formation, or acetyl phosphate production. Coenzyme A can be measured using thiol-reactive colorimetric methods, whereas acetyl-CoA and acetyl phosphate can be detected using enzyme-coupled or chromatographic methods. Because the PTA reaction is reversible, the reaction direction must be clearly defined during measurement. When comparing different samples, substrate concentrations, pH, temperature, reaction time, and protein quantification methods should be standardized.

7.2 Acetate Kinase Activity Assays

ACK activity can be measured by detecting ATP or acetate production in the forward reaction or by detecting ADP or acetyl phosphate formation in the reverse reaction. ATP and ADP are commonly measured using luminescent, colorimetric, or enzyme-coupled methods. ATPases, phosphatases, and other kinases in crude enzyme extracts may interfere with the assay. Therefore, enzyme-free, substrate-free, and heat-inactivated controls should be included, and enzyme activity should be measured during the initial linear phase of the reaction.

7.3 Acetyl-CoA Synthetase Activity Assays

ACS activity can be evaluated by measuring acetyl-CoA, AMP, pyrophosphate, or coenzyme A consumption. Enzyme-coupled assays are suitable for continuous kinetic measurements, whereas liquid chromatography and mass spectrometry can directly confirm the reaction products. ACS is a multisubstrate enzyme. Kinetic analysis should separately vary acetate, ATP, and coenzyme A concentrations. A single substrate concentration cannot fully characterize substrate affinity and catalytic efficiency.

7.4 Acetate Content Determination

Acetate in culture media can be measured using enzymatic assay kits, high-performance liquid chromatography, ion chromatography, or gas chromatography. Enzymatic methods are suitable for routine batch measurements, whereas chromatographic methods can simultaneously distinguish acetate, formate, lactate, succinate, and other organic acids. Cells should be removed by centrifugation or filtration before analysis, and dilution factors and sampling times should be recorded. Dark-colored, high-sugar, or high-salt fermentation broths may interfere with colorimetric assays and require matrix blanks and spike-recovery controls.

7.5 Measurement of Acetyl-CoA and Acetyl Phosphate

Acetyl-CoA can be measured using enzyme-coupled methods, liquid chromatography, or mass spectrometry. Coenzyme A thioesters are prone to degradation, so metabolism should be rapidly quenched after sampling and extraction completed under low-temperature conditions. Acetyl phosphate has low chemical stability and readily undergoes hydrolysis or acetyl-group transfer. The interval between sampling and extraction should therefore be minimized, and acetyl phosphate levels should not be inferred solely from pta or ackA expression.

7.6 Evaluation of Energy Status and Carbon Flux

ATP, ADP, and AMP contents can be used to evaluate cellular energy status, whereas the NADH/NAD⁺ ratio reflects the relationship between acetate metabolism and respiratory or fermentative states. Carbon-source consumption, cell biomass, and target-product formation should also be measured when evaluating acetate metabolism. A decrease in endpoint acetate concentration may result from reduced acetate production, enhanced acetate reutilization, or inhibited cell growth. Establishing a time series across the culture process helps distinguish among these mechanisms.

7.7 Comparison of Common Detection Indicators

Table 3 Common Methods for Measuring Acetate Metabolic Enzyme Activity and Metabolic Products

8 Products for Key Acetate Metabolic Enzymes, Reaction Substrates, and Metabolic Evaluation

Table 4 Enzymes and Activity Assay Kits Related to Acetate Production and Utilization

Table 5 Substrates, Coenzymes, and Key Intermediate Products for Acetate Metabolism

Table 6 Products for Evaluating Downstream Acetyl-CoA Flux, Energy Status, and Redox State

Microbial acetate metabolism simultaneously contributes to ATP generation, acetyl-CoA distribution, coenzyme A regeneration, and environmental adaptation. The PTA-ACK pathway is suited to rapid excretion of excess carbon and ATP recovery, whereas ACS is responsible for high-affinity acetate activation.

For more related articles, please see below:

[1] Acetyl-Group Metabolic Enzyme Network and In Vitro Enzymology Analysis