Overflow metabolism refers to the production of seemingly wasteful by-products by cells during growth on glycolytic substrates such as glucose, even when oxygen is abundant. Acetate overflow is a metabolic process in which a large portion of the carbon is absorbed as glucose into Escherichia coli cells and it is catabolized and expelled into the medium as acetate. Previous studies have shown that mutants for the acetate overflow pathway enzymes acetate kinase (AckA) and phosphoracetyltransferase (Pta) demonstrated high diauxic growth after glucose depletion in Escherichia coli (Shimada and Tanaka, 2016).
A new study conducted by Tokyo Institute of Technology researchers and led by Professor Kan Tanaka discovered a new role for acetate overflow in E. coli (Shimada et al., 2021). Bacterial cells absorb a large amount of carbon when glucose is catabolized via glycolysis and expelled as acetate through acetate overflow. Several theories have been proposed to explain the physiological significance of this acetate overflow because it appears to be a waste of carbon resources. The relevance of acetate overflow during steady-state growth was well explained by the proteome allocation theory, as the proteomic cost of energy synthesis via respiration surpasses that by fermentation. The research team also explained that acetate overflow is also causes continual growth after glucose depletion in the presence of excess amino acids. Acetate overflow reduces pyruvate concentration and aids in the supply of HS-CoA.
Shimada and colleagues investigated the underlying mechanism in the pta mutant. Pyruvate buildup caused a decrease in glyoxylate shunt enzymes and an increase in pyruvate dehydrogenase complex subunits. These modifications were thought to be the cause of the deficiency since overexpressing PdhR (pyruvate-sensing transcription factor) or deleting iclR (gene encoding pyruvate- and glyoxylate-sensing transcription factor) reduced the pta mutant’s growth lag after glucose depletion. Because the pta mutant had lower levels of coenzyme A (HS-CoA), the growth inhibition following glucose depletion could be explained by a lack of HS-CoA in the cell. The findings reveal the importance of acetate overflow in metabolic regulation, which could have significant biotechnological effects.
Increased pyruvate levels in acetate overflow mutants cause a rise or decrease in the HS-CoA-consuming PDHc or the HS-CoA-generating glyoxylate shunt enzyme, malate synthase A (AceB), respectively. It also contributes to the cell’s HS-CoA availability. Because Pta is the enzyme that releases HS-CoA, HS-CoA should be restricted even more in the pta mutant than in the ackA mutant. After a significant lag period, post-glucose growth is finally regained. It suggests that acetate overflow is required for the metabolic shift to the TCA, PEP-glyoxylate cycles and a gluconeogenesis-centered metabolic state. The glyoxylate shunt also plays a role in gluconeogenesis, which is necessary for growth after glucose shortage.
Previous studies have proposed that HS-CoA restriction in vivo causes coordinated and competing activation of PDHc and OGDHc. The authors believe that after glucose deprivation, a comparable competition for HS-CoA occurs for the TCA cycle enzyme OGDHc activation that suppresses the growth. Both PDHc and OGDHc are large complex enzymes with identical E1, E2, and E3 subunit compositions. The E1 and E2 subunits are homologous but distinct proteins, while the common E3 component (Lpd) is found in both complexes. They perform similar reactions such as the formation of acyl-CoA thioesters, decarboxylation of 2-ketoacids, and reduction of pyridine dinucleotide NAD+ to NADH. Cofactors essential for these enzymes: lipoic acid, NAD+, HS-CoA, and thiamine pyrophosphate are common for both reactions. These two enzyme activities could be coordinated through the management of these common subunits and cofactors.
The researchers successfully showed a new role for acetate overflow in the metabolic state transition after glucose depletion in the presence of excess amino acids in E. coli. Higher levels of pyruvate cause reduction of glyoxylate shunt enzymes and stop the availability of HS-CoA. Overflow of acetate stops pyruvate accumulation and limits its impact. The current findings provide important insights into the underlying mechanism and are valuable to understand the overflow of acetate process and it advances our understanding of various biotechnological applications involving related bacteria.
Tomohiro Shimada and Kan Tanaka. “Use of a bacterial luciferase monitoring system to estimate real-time dynamics of intracellular metabolism in Escherichia coli.” Appl Environ Microb. 82, 5960-5968 (2016). DOI: 10.1128/aem.01400-16
Tomohiro Shimada, Kohta Nakazawa, Tomoyuki Tachikawa, Natsumi Saito, Tatsuya Niwa, Hideki Taguchi and Kan Tanaka. “Acetate overflow metabolism regulates a major metabolic shift after glucose depletion in Escherichia coli.” FEBS Letters 595, 2047-2056 (2021). DOI:10.1002/1873-3468.14151