ackA Mutants Research Articles

Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21.

Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB1′ and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.

The Impact of ackA, pta, and ackA-pta Mutations on Growth, Gene Expression and Protein Acetylation in Escherichia coli K-12

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.

Identification and characterization of AckA-dependent protein acetylation in Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the causative agent of gonorrhea, has a number of factors known to contribute to pathogenesis; however, a full understanding of these processes and their regulation has proven to be elusive. Post-translational modifications (PTMs) of bacterial proteins are now recognized as one mechanism of protein regulation. In the present study, Western blot analyses, with an anti-acetyl-lysine antibody, indicated that a large number of gonococcal proteins are post-translationally modified. Previous work has shown that Nε-lysine acetylation can occur non-enzymatically with acetyl-phosphate (AcP) as the acetyl donor. In the current study, an acetate kinase mutant (1291ackA), which accumulates AcP, was generated in N. gonorrhoeae. Broth cultures of N. gonorrhoeae 1291wt and 1291ackA were grown, proteins extracted and digested, and peptides containing acetylated-lysines (K-acetyl) were affinity-enriched from both strains. Mass spectrometric analyses of these samples identified a total of 2686 unique acetylation sites. Label-free relative quantitation of the K-acetyl peptides derived from the ackA and wild-type (wt) strains demonstrated that 109 acetylation sites had an ackA/wt ratio>2 and p-values <0.05 in at least 2/3 of the biological replicates and were designated as “AckA-dependent”. Regulated K-acetyl sites were found in ribosomal proteins, central metabolism proteins, iron acquisition and regulation proteins, pilus assembly and regulation proteins, and a two-component response regulator. Since AckA is part of a metabolic pathway, comparative growth studies of the ackA mutant and wt strains were performed. The mutant showed a growth defect under aerobic conditions, an inability to grow anaerobically, and a defect in biofilm maturation. In conclusion, the current study identified AckA-dependent acetylation sites in N. gonorrhoeae and determined that these sites are found in a diverse group of proteins. This work lays the foundation for future studies focusing on specific acetylation sites that may have relevance in gonococcal pathogenesis and metabolism.

Redox Imbalance Underlies the Fitness Defect Associated with Inactivation of the Pta-AckA Pathway in Staphylococcus aureus.

The phosphotransacetylase-acetate kinase (Pta-AckA) pathway is thought to be a vital ATP generating pathway for Staphylococcus aureus. Disruption of the Pta-AckA pathway during overflow metabolism causes significant reduction in growth rate and viability, albeit not due to intracellular ATP depletion. Here, we demonstrate that toxicity associated with inactivation of the Pta-AckA pathway resulted from an altered intracellular redox environment. Growth of the pta and ackA mutants under anaerobic conditions partially restored cell viability. NMR metabolomics analyses and (13)C6-glucose metabolism tracing experiments revealed the activity of multiple pathways that promote redox (NADH/NAD(+)) turnover to be enhanced in the pta and ackA mutants during anaerobic growth. Restoration of redox homeostasis in the pta mutant by overexpressing l- lactate dehydrogenase partially restored its viability under aerobic conditions. Together, our findings suggest that during overflow metabolism, the Pta-AckA pathway plays a critical role in preventing cell viability defects by promoting intracellular redox homeostasis.

Impact of deletion of the genes encoding acetate kinase on production of L-tryptophan by Escherichia coli

The production of L-tryptophan was increased by reducing acetate accumulation through a decrease in acetate kinase activity by gene deletion. The effects of disruption of the genes for acetate kinase (ackA) and an enzyme with propionate/acetate kinase activity (tdcD) on L-tryptophan production were investigated. The ackA and/or tdcD deletion mutants accumulated less acetate and more L-tryptophan than the parental strain. Furthermore, the production of L-tryptophan obtained with ackA-tdcD mutant were more than the mutants with a single deletion of ackA or tdcD, while higher production of L-tryptophan and lower concentration of acetate were accumulated in the ackA mutant than the mutant with a lesion in tdcD. In L-tryptophan fed-batch fermentation using the ackA-tdcD mutant, the excretion of acetate was reduced to 1.22 g/L, a 21.79 % reduction compared with the parental strain, and the production of L-tryptophan and glucose conversion rate were increased to 52.5 g/L and 47.9 g/L, respectively, which represented 6.49 % and 10.88 % increases compared with the parental strain, and the glucose conversion rate reached a high level of 21.2 %, which was 8.16 % higher than the parental strain. In addition, the metabolic flux analysis of TRTH and TRTHAT indicated that the carbon flux through EMP was decreased by 8.37 % and the carbon flux through PP was increased by 57.03 % in TRTHAT compared with TRTH. The flux of acetate and tryptophan formation of TRTHAT were 5.2 % and 17.3 %, which were 3.67-times lower and 1.75-times higher than these of TRTH, respectively.

Production of 4-hydroxybutyric acid by metabolically engineered Mannheimia succiniciproducens and its conversion to γ-butyrolactone by acid treatment

γ-Butyrolactone (GBL) is an important four carbon (C4) chemical, which has a wide range of industrial applications. GBL can be produced by acid treatment of 4-hydroxybutyric acid (4-HB), which is a derivative of succinic acid. Heterologous metabolic pathways were designed and established in succinic acid overproducing Mannheimia succiniciproducens LPK7 (ldhA pflD pta ackA mutant) by the introduction of heterologous genes that encode succinyl-CoA synthetase, CoA-dependent succinate semialdehyde dehydrogenase, and either 4-hydroxybutyrate dehydrogenase in LPK7 (p3S4CD) or succinate semialdehyde reductase in LPK7 (p3SYCD). Fed-batch cultures of LPK7 (p3S4CD) and LPK7 (p3SYCD) resulted in the production of 6.37 and 6.34g/L of 4-HB (molar yields of 0.143 and 0.139), respectively. Finally, GBL was produced by acid treatment of the 4-HB obtained from the fermentation broth with molar yield of 0.673. This study demonstrates that 4-HB, and potentially other four carbon platform chemicals, can be produced by the engineered rumen bacterium M. succiniciproducens.

Pyruvate Formate Lyase and Acetate Kinase Are Essential for Anaerobic Growth ofEscherichia colion Xylose

During anaerobic growth of bacteria, organic intermediates of metabolism, such as pyruvate or its derivatives, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate-level phosphorylation. In Escherichia coli, conversion of glucose to pyruvate yields 2 net ATPs, while metabolism of a pentose, such as xylose, to pyruvate only yields 0.67 net ATP per xylose due to the need for one (each) ATP for xylose transport and xylulose phosphorylation. During fermentative growth, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP from two pyruvates (one hexose equivalent) while still maintaining the overall redox balance. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose. An E. coli pfl mutant lacking pyruvate formate lyase cannot convert pyruvate to acetyl coenzyme A, the required precursor for acetate and ethanol production, and could not produce this additional ATP. E. coli pfl mutants failed to grow under anaerobic conditions in xylose minimal medium without any negative effect on their survival or aerobic growth. An ackA mutant, lacking the ability to generate ATP from acetyl phosphate, also failed to grow in xylose minimal medium under anaerobic conditions, confirming the need for the ATP produced by acetate kinase for anaerobic growth on xylose. Since arabinose transport by AraE, the low-affinity, high-capacity, arabinose/H+ symport, conserves the ATP expended in pentose transport by the ABC transporter, both pfl and ackA mutants grew anaerobically with arabinose. AraE-based xylose transport, achieved after constitutively expressing araE, also supported the growth of the pfl mutant in xylose minimal medium. These results suggest that a net ATP yield of 0.67 per pentose is only enough to provide for maintenance energy but not enough to support growth of E. coli in minimal medium. Thus, pyruvate formate lyase and acetate kinase are essential for anaerobic growth of E. coli on xylose due to energetic constraints.

An analysis of multifactorial influences on the transcriptional control of ompF and ompC porin expression under nutrient limitation.

Expression of the major outer-membrane porins in Escherichia coli is transcriptionally controlled during nutrient limitation. Expression of ompF was more than 40-fold higher under glucose limitation than under nitrogen (ammonia) limitation in chemostat cultures at the same growth rate. In contrast, ompC expression was higher under N limitation. The basis of regulation by nutrient limitation was investigated using mutations affecting expression of porin genes. The influence of cyaA, rpoS, ackA and pta, as well as the two-component envZ-ompR system, was studied under glucose and N limitation in chemostat cultures. A major contributor to low ompF expression under N limitation was negative control by the RpoS sigma factor. RpoS levels were high under N limitation and loss of RpoS resulted in a 19-fold increase in ompF transcription, but little change was observed with ompC. Lack of RpoS under glucose limitation had a lesser stimulatory effect on ompF expression. Porin production was minimally dependent on EnvZ under N limitation due to OmpR phosphorylation by acetyl phosphate. Evidence obtained with pta and ackA mutants suggested that the acetyl phosphate level also regulates porins independently and indirectly via RpoS and other pathways. pta-envZ double mutants had a residual level of porin transcription, implicating alternative means of OmpR phosphorylation under nutrient limitation. Another critical factor in regulation was the level of cAMP, as a cyaA mutant hardly expressed ompF under glucose limitation but boosted ompC. In addition, the role of DNA-binding proteins encoded by hns and himA was tested under glucose limitation: the hns mutation reduced the glucose-limitation peak, but the himA mutation suppressed the hns effect, suggesting a complex web of interrelationships between the DNA-binding proteins. Indeed, multiple inputs and no single regulator were responsible for the high peak of ompF expression under glucose limitation.

Effects of mutations in acetate metabolism on high-cell-density growth of Escherichia coli

To study the role played by acetate metabolism during high-cell-density growth of Escherichia coli cells, we constructed isogenic null mutants of strain W3100 deficient for several genes involved either in acetate metabolism or the transition to stationary phase. We grew these strains under identical fed-batch conditions to the highest cell densities achievable in 8 h using a predictive-plus-feedback-controlled computer algorithm that maintained glucose at a set-point of 0.5 g/l, as previously described. Wild-type strains, as well as mutants lacking the ss subunit of RNA polymerase (rpoS), grew reproducibly to high cell densities (44–50 g/l dry cell weights, DCWs). In contrast, a strain lacking acetate kinase (ackA) failed to reach densities greater than 8 g/l. Strains lacking other acetate metabolism genes (pta, acs, poxB, iclR, and fadR) achieved only medium cell densities (15–21 g/l DCWs). Complementation of either the acs or the ackA mutant restored wild-type high-cell-density growth. On a dry weight basis, poxB and fadR strains produced approximately threefold more acetate than did the wild-type strain. In contrast, the pta, acs, or rpoS strains produced significantly less acetate per cell dry weight than did the wild-type strain. Our results show that acetate metabolism plays a critical role during growth of E. coli cultures to high cell densities. They also demonstrate that cells do not require the ss regulon to grow to high cell densities, at least not under the conditions tested. Journal of Industrial Microbiology & Biotechnology (2000) 24, 421–430.

Involvement of carbon source and acetyl phosphate in the external-pH-dependent expression of porin genes in Escherichia coli.

The porin composition of the Escherichia coli cell envelope was analyzed during growth at different external pHs (pHo) as a function of the acetyl phosphate (AcP) level (DeltaackA pta or ackA mutant, pyruvate or glucose as the carbon source) in the presence or absence of EnvZ. Our results indicate that the AcP level is influenced by the pHo, leading to modulation of the amount of OmpR-P and subsequent pHo-dependent expression of ompF and ompC. We also propose the existence of a specific signal, independent of EnvZ and AcP, leading to OmpR phosphorylation in response to pyruvate.

The glucose‐starvation stimulon of Escherichia coli: induced and repressed synthesis of enzymes of central metabolic pathways and role of acetyl phosphate in gene expression and starvation survival

Proteins of the glucose-starvation stimulon were identified by using two-dimensional gel electrophoresis and the gene-protein database of Escherichia coli. Members of this stimulon included enzymes of the Embden-Meyerhof-Parnas (EMP) pathway, phosphotransacetylase (Pta) and acetate kinase (AckA) of the acetyl phosphate/acetate production pathway, and formate transacetylase. The synthesis of these enzymes was found to be induced concomitantly with the decreased synthesis of enzymes of the Krebs cycle. Thus, the modulation in the synthesis of specific proteins during aerobic glucose starvation is, in part, similar to the response of cells shifted to anaerobiosis. These modulations suggest that the glucose-starved cell increases the relative flow of carbon through the Pta-AckA pathway. Indeed, the ability to synthesize acetyl phosphate, an intermediate of the pathway, appears to be indispensable for glucose-starved cells as pta and pta-ackA double mutants were found to be impaired in their ability to survive glucose starvation. The survival characteristics of ackA mutants and the wild-type parent were indistinguishable. Moreover, the pta mutant failed to induce several proteins of the glucose-starvation stimulon.