Year-end Sale % OFF 
Abstract
Acetate is a characteristic by-product of Escherichia coli K-12 growing in batch cultures with glucose, both under aerobic as well as anaerobic conditions. While the reason underlying aerobic acetate production is still under discussion, during anaerobic growth acetate production is important for ATP generation by substrate level phosphorylation. Under both conditions, acetate is produced by a pathway consisting of the enzyme phosphate acetyltransferase (Pta) producing acetyl-phosphate from acetyl-coenzyme A, and of the enzyme acetate kinase (AckA) producing acetate from acetyl-phosphate, a reaction that is coupled to the production of ATP. Mutants in the AckA-Pta pathway differ from each other in the potential to produce and accumulate acetyl-phosphate. In the publication at hand, we investigated different mutants in the acetate pathway, both under aerobic as well as anaerobic conditions. While under aerobic conditions only small changes in growth rate were observed, all acetate mutants showed severe reduction in growth rate and changes in the by-product pattern during anaerobic growth. The AckA– mutant showed the most severe growth defect. The glucose uptake rate and the ATP concentration were strongly reduced in this strain. This mutant exhibited also changes in gene expression. In this strain, the atoDAEB operon was significantly upregulated under anaerobic conditions hinting to the production of acetoacetate. During anaerobic growth, protein acetylation increased significantly in the ackA mutant. Acetylation of several enzymes of glycolysis and central metabolism, of aspartate carbamoyl transferase, methionine synthase, catalase and of proteins involved in translation was increased. Supplementation of methionine and uracil eliminated the additional growth defect of the ackA mutant. The data show that anaerobic, fermentative growth of mutants in the AckA-Pta pathway is reduced but still possible. Growth reduction can be explained by the lack of an important ATP generating pathway of mixed acid fermentation. An ackA deletion mutant is more severely impaired than pta or ackA-pta deletion mutants. This is most probably due to the production of acetyl-P in the ackA mutant, leading to increased protein acetylation.
Highlights
In Escherichia coli K12 acetate is a major product of metabolism under both aerobic and anaerobic growth conditions (Figure 1) (Wolfe, 2005; Bernal et al, 2016)
To analyze the fermentative growth of ackA, pta, and ackA-pta deletion mutants, we investigated growth of isogenic mutants (Table 1) in defined medium with glucose as sole carbon source All three mutants showed significantly reduced growth rates and a lower biomass yield (Figure 2 and Table 2)
To check if PoxB is involved in acetate production in the ackA mutant, we constructed the poxB ackA deletion mutant KBM16121 (Table 1)
Summary
In Escherichia coli K12 acetate is a major product of metabolism under both aerobic and anaerobic growth conditions (Figure 1) (Wolfe, 2005; Bernal et al, 2016). Acetate is predominantly produced by a pathway originating from acetyl-coenzyme A (acetyl-CoA) that is catalyzed by the enzymes phosphate acetyltransferase (encoded by pta) and acetate kinase (encoded by ackA) (Brown et al, 1977). Acetate represents a major product of mixed acid fermentation (Sawers and Clark, 2004). Pyruvate produced by the glycolytic reactions is converted into acetyl-CoA and formate by the enzyme pyruvate formate lyase (PFL) (Sawers and Clark, 2004). ATP production by the AckA-Pta pathway is important for growth under fermentative conditions. PoxB is a common target in metabolic engineering approaches, too
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
