scholarly journals Impact of CO2/HCO3– Availability on Anaplerotic Flux in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Aileen Krüger ◽  
Johanna Wiechert ◽  
Cornelia Gätgens ◽  
Tino Polen ◽  
Regina Mahr ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Carboxylase ◽  
Metabolic Flux ◽  
Pyruvate Dehydrogenase Complex ◽  
Lag Phase ◽  
Production Rates ◽  
Content Type ◽  
The Impact ◽  
Dehydrogenase Complex

ABSTRACT The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative decarboxylation of pyruvate, yielding acetyl coenzyme A (acetyl-CoA) and CO2. The PDHC-deficient Corynebacterium glutamicum ΔaceE strain therefore lacks an important decarboxylation step in its central metabolism. Additional inactivation of pyc, encoding pyruvate carboxylase, resulted in a >15-h lag phase in the presence of glucose, while no growth defect was observed on gluconeogenetic substrates, such as acetate. Growth was successfully restored by deletion of ptsG, encoding the glucose-specific permease of the phosphotransferase system (PTS), thereby linking the observed phenotype to the increased sensitivity of the ΔaceE Δpyc strain to glucose catabolism. In this work, the ΔaceE Δpyc strain was used to systematically study the impact of perturbations of the intracellular CO2/HCO3– pool on growth and anaplerotic flux. Remarkably, all measures leading to enhanced CO2/HCO3– levels, such as external addition of HCO3–, increasing the pH, or rerouting metabolic flux via the pentose phosphate pathway, at least partially eliminated the lag phase of the ΔaceE Δpyc strain on glucose medium. In accordance with these results, inactivation of the urease enzyme, lowering the intracellular CO2/HCO3– pool, led to an even longer lag phase, accompanied by the excretion of l-valine and l-alanine. Transcriptome analysis, as well as an adaptive laboratory evolution experiment with the ΔaceE Δpyc strain, revealed the reduction of glucose uptake as a key adaptive measure to enhance growth on glucose-acetate mixtures. Taken together, our results highlight the significant impact of the intracellular CO2/HCO3– pool on metabolic flux distribution, which becomes especially evident in engineered strains exhibiting low endogenous CO2 production rates, as exemplified by PDHC-deficient strains. IMPORTANCE CO2 is a ubiquitous product of cellular metabolism and an essential substrate for carboxylation reactions. The pyruvate dehydrogenase complex (PDHC) catalyzes a central metabolic reaction contributing to the intracellular CO2/HCO3– pool in many organisms. In this study, we used a PDHC-deficient strain of Corynebacterium glutamicum, which additionally lacked pyruvate carboxylase (ΔaceE Δpyc). This strain featured a >15-h lag phase during growth on glucose-acetate mixtures. We used this strain to systematically assess the impact of alterations in the intracellular CO2/HCO3– pool on growth in glucose-acetate medium. Remarkably, all measures enhancing CO2/HCO3– levels successfully restored growth. These results emphasize the strong impact of the intracellular CO2/HCO3– pool on metabolic flux, especially in strains exhibiting low endogenous CO2 production rates.

scholarly journals The impact of CO2/HCO3-availability on anaplerotic flux in PDHC-deficientCorynebacterium glutamicumstrains

2019 ◽  
Author(s):  
Aileen Krüger ◽  
Johanna Wiechert ◽  
Cornelia Gätgens ◽  
Tino Polen ◽  
Regina Mahr ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Carboxylase ◽  
Metabolic Flux ◽  
Pyruvate Dehydrogenase Complex ◽  
Lag Phase ◽  
Metabolic Reaction ◽  
Production Rates ◽  
The Impact ◽  
Dehydrogenase Complex

AbstractThe pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative decarboxylation of pyruvate yielding acetyl-CoA and CO2. The PDHC-deficientCorynebacterium glutamicumstrain ΔaceEis therefore lacking an important decarboxylation step in central metabolism. Additional inactivation ofpyc, encoding pyruvate carboxylase, resulted in a >15 hour lag phase in the presence of glucose, while no growth defect was observed on gluconeogenetic substrates like acetate. Growth was successfully restored by deletion ofptsGencoding the glucose-specific permease of the PTS system, thereby linking the observed phenotype to the increased sensitivity of strain ΔaceEΔpycto glucose catabolism. In the following, strain ΔaceEΔpycwas used to systematically study the impact of perturbations of the intracellular CO2/HCO3-pool on growth and anaplerotic flux. Remarkably, all measures leading to enhanced CO2/HCO3-levels, such as external addition of HCO3-, increasing the pH, or rerouting metabolic flux via pentose phosphate pathway, at least partially eliminated the lag phase of strain ΔaceEΔpycon glucose medium. In accordance, inactivation of the urease enzyme, lowering the intracellular CO2/HCO3-pool, led to an even longer lag phase accompanied with the excretion of L-valine and L-alanine. Transcriptome analysis as well as an adaptive laboratory evolution experiment of strain ΔaceEΔpycrevealed the reduction of glucose uptake as a key adaptive measure to enhance growth on glucose/acetate mixtures. Altogether, our results highlight the significant impact of the intracellular CO2/HCO3-pool on metabolic flux distribution, which becomes especially evident in engineered strains suffering from low endogenous CO2production rates as exemplified by PDHC-deficient strains.ImportanceCO2is a ubiquitous product of cellular metabolism and an essential substrate for carboxylation reactions. The pyruvate dehydrogenase complex (PDHC) catalyzes a central metabolic reaction contributing to the intracellular CO2/HCO3-pool in many organisms. In this study, we used a PDHC-deficient strain ofCorynebacterium glutamicum, which was additionally lacking pyruvate carboxylase (ΔaceEΔpyc). This strain featured a >15 h lag phase during growth on glucose-acetate mixtures. We used this strain to systematically assess the impact of alterations in the intracellular CO2/HCO3-pool on growth on glucose-containing medium. Remarkably, all measures enhancing the CO2/HCO3-levels successfully restored growth emphasizing the strong impact of the intracellular CO2/HCO3-pool on metabolic flux especially in strains suffering from low endogenous CO2production rates.

scholarly journals Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine, l-Valine, and 2-Ketoisovalerate

2013 ◽  
Vol 79 (18) ◽  
pp. 5566-5575 ◽  
Author(s):  
Jens Buchholz ◽  
Andreas Schwentner ◽  
Britta Brunnenkan ◽  
Christina Gabris ◽  
Simon Grimm ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Fed Batch ◽  
Complex Activity ◽  
Gene Encoding ◽  
Content Type ◽  
Genes Encoding ◽  
Cell Densities ◽  
Dehydrogenase Complex

ABSTRACTExchange of the nativeCorynebacterium glutamicumpromoter of theaceEgene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutateddapApromoter variants led to a series ofC. glutamicumstrains with gradually reduced growth rates and PDHC activities. Upon overexpression of thel-valine biosynthetic genesilvBNCE, all strains producedl-valine. Among these strains,C. glutamicum aceEA16 (pJC4ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of thepqoandppcgenes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities,C. glutamicum aceEA16 Δpqo Δppc(pJC4ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter)l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression ofilvBNCDinstead ofilvBNCEtransformed thel-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with aYP/Sof 0.24 mol per mol of glucose and aQPof 6.9 mM per h [0.8 g/(liter × h)]. The replacement of theaceEpromoter by thedapA-A16 promoter in the twoC. glutamicuml-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate thatC. glutamicumstrains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

scholarly journals Comparative13C Metabolic Flux Analysis of Pyruvate Dehydrogenase Complex-Deficient, l-Valine-Producing Corynebacterium glutamicum

2011 ◽  
Vol 77 (18) ◽  
pp. 6644-6652 ◽  
Author(s):  
Tobias Bartek ◽  
Bastian Blombach ◽  
Siegmund Lang ◽  
Bernhard J. Eikmanns ◽  
Wolfgang Wiechert ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Pyruvate Dehydrogenase Complex ◽  
Pentose Phosphate ◽  
Strong Increase ◽  
Flux Analysis ◽  
Wild Type ◽  
Content Type ◽  
Split Ratio

ABSTRACTl-Valine can be formed successfully usingC. glutamicumstrains missing an active pyruvate dehydrogenase enzyme complex (PDHC). Wild-typeC. glutamicumand four PDHC-deficient strains were compared by13C metabolic flux analysis, especially focusing on the split ratio between glycolysis and the pentose phosphate pathway (PPP). Compared to the wild type, showing a carbon flux of 69% ± 14% through the PPP, a strong increase in the PPP flux was observed in PDHC-deficient strains with a maximum of 113% ± 22%. The shift in the split ratio can be explained by an increased demand of NADPH forl-valine formation. In accordance, the introduction of theEscherichia colitranshydrogenase PntAB, catalyzing the reversible conversion of NADH to NADPH, into anl-valine-producingC. glutamicumstrain caused the PPP flux to decrease to 57% ± 6%, which is below the wild-type split ratio. Hence, transhydrogenase activity offers an alternative perspective for sufficient NADPH supply, which is relevant for most amino acid production systems. Moreover, as demonstrated forl-valine, this bypass leads to a significant increase of product yield due to a concurrent reduction in carbon dioxide formation via the PPP.

scholarly journals Pyruvate production by Escherichia coli using pyruvate dehydrogenase variants

2021 ◽  
Author(s):  
W. Chris Moxley ◽  
Mark A. Eiteman
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Carbon Flux ◽  
Metabolic Flux ◽  
Pyruvate Dehydrogenase Complex ◽  
Cost Effective ◽  
Specific Productivity ◽  
Synthesis Methods ◽  
Additional Tool ◽  
Dehydrogenase Complex

Altering metabolic flux at a key branchpoint in metabolism has commonly been accomplished through gene knockouts or by modulating gene expression. An alternative approach to direct metabolic flux preferentially toward a product is decreasing the activity of a key enzyme through protein engineering. In Escherichia coli, pyruvate can accumulate from glucose when carbon flux through the pyruvate dehydrogenase complex is suppressed. Based on this principle, 16 chromosomally expressed AceE variants were constructed in E. coli C and compared for growth rate and pyruvate accumulation using glucose as the sole carbon source. To prevent conversion of pyruvate to other products, the strains also contained deletions in two nonessential pathways: lactate dehydrogenase (ldhA) and pyruvate oxidase (poxB). The effect of deleting phosphoenolpyruvate synthase (ppsA) on pyruvate assimilation was also examined. The best pyruvate-accumulating strains were examined in controlled batch and continuous processes. In a nitrogen-limited chemostat process at steady-state growth rates of 0.15 – 0.28 h−1, an engineered strain expressing the AceE[H106V] variant accumulated pyruvate at a yield of 0.59-0.66 g pyruvate/g glucose with a specific productivity of 0.78 – 0.92 g pyruvate/g cells·h. These results provide proof-of-concept that pyruvate dehydrogenase complex variants can effectively shift carbon flux away from central carbon metabolism to allow pyruvate accumulation. This approach can potentially be applied to other key enzymes in metabolism to direct carbon toward a biochemical product. Importance Microbial production of biochemicals from renewable resources has become an efficient and cost-effective alternative to traditional chemical synthesis methods. Metabolic engineering tools are important for optimizing a process to perform at an economically feasible level. This study describes an additional tool to modify central metabolism and direct metabolic flux to a product. We have shown that variants of the pyruvate dehydrogenase complex can direct metabolic flux away from cell growth to increase pyruvate production in Escherichia coli. This approach could be paired with existing strategies to optimize metabolism and create industrially relevant and economically feasible processes.

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