Figure 7. Optimization of the E. A Effect of host strains on PHB production from ethanol. C shows a representative result from two independent experiments. The chaperone DnaK was proved to protect the mutant AdhE against metal-catalyzed oxidation and to improve the aerobic growth on ethanol of E. Then, the strain Q was subjected to fed-batch fermentation to test the feasibility of PHB production from ethanol in large scale.
After h fermentation, the strain Q produced It was worth noting that the byproduct formate was accumulated to a concentration of To test whether ethanol is suitable for production of other acetyl-CoA derived chemicals, the biosynthetic pathways for PG and 3HP from glucose, ethanol and acetate were constructed in E.
The theoretical yields of 3HP and PG from various carbon sources were calculated, and those from ethanol were the highest Figures 8A,B. Figure 8. A 3HP biosynthetic pathway and theoretical yield from different carbon sources.
B PG biosynthetic pathway and theoretical yield from different carbon sources. C Cell density and 3HP production when grown with glucose, ethanol and acetate as sole carbon source.
D Cell density and PG production when grown with glucose, ethanol and acetate as sole carbon source. After cultivation in shaking flasks, Q strain grown on ethanol produced 0.
These results demonstrate that ethanol can be used as carbon source for production of acetyl-CoA derived chemicals besides PHB. Acetyl-CoA is a fundamental metabolite in bacterial central metabolic pathways, and also a precursor for biosynthesis of large number of materials and chemicals Martin et al. A series of acetyl-CoA derived chemical and material have been successfully produced by engineered E.
Conversion of glucose into acetyl-CoA presents low atomic economy due to the release of CO 2 , leading to decrease of theoretical production yield, titer and productivity of target product Chae et al. To improve the atomic economy of biosynthesis process, fatty acids were tested as an alternative carbon source Liu et al. Although the production and yield of target chemical were increased significantly Liu et al. Several synthetic pathways for acetyl-CoA from one-carbon substrates have been constructed in E.
In this study, E. Furthermore, metabolome analysis was carried out to discover the differences between metabolism of glucose, ethanol and acetate in engineered E. All these results suggested that ethanol may be a suitable carbon source for production of acetyl-CoA derived bioproducts. Firstly, ethanol is an ordinary and inexpensive commodity chemical.
Secondly, the conversion of ethanol into acetyl-CoA presents high atomic economy, in addition to the generation of NADH. Therefore, the theoretical yield of target chemical from ethanol is much higher than those from glucose and acetate Figures 5A , 7A,B. Furthermore, ethanol has a higher energy density. If oxidized completely to CO 2 and H 2 O in bacteria, 0. Moreover, E. As glucose is the favorite carbon source of E. The assimilation of ethanol was neither as fast as glucose to accumulate by-products, nor as slow as acetate to retard the bacterial growth, helping bacteria reach a balance between growth and production.
Additionally, the nature of ethanol, such as low toxicity and high water-solubility, makes it friendly to the bacterial cultivation process. Although our study showed the feasibility of ethanol to support E.
The ethanol utilizing gene adhE mut was carried by a plasmid vector, leading to addition of antibiotic into medium and increasing of production cost. Besides that, the strain performance may be affected by the strain instability due to plasmid loss. This problem can be dissolved by the integration of adhE mut gene into bacterial chromosome. Furthermore, the PHB yield was 0. It is necessary to carry out further development to achieve a higher production and yield from ethanol.
Compared with glucose and acetate, the strains grown on ethanol presented the highest production and yield, and metabolome analysis revealed the reasons of high yield from ethanol. All these results demonstrate that ethanol is a putative carbon source for production of acetyl-CoA derived bioproducts. GZ and MX designed the study. SS and GZ wrote the manuscript. All authors read and approved the final manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Dr. This manuscript has been released as a pre-print at Research Square Sun et al. Aldor, I. Metabolic engineering of poly 3-hydroxybutyrate-cohydroxyvalerate composition in recombinant Salmonella enterica serovar typhimurium.
Bates, D. Self-assembly and catalytic activity of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. Nature , — Brandl, H. Bunch, P. The ldhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli. Microbiology Pt 1 , — Cao, Y. Improved phloroglucinol production by metabolically engineered Escherichia coli.
Increasing unsaturated fatty acid contents in Escherichia coli by coexpression of three different genes. Chae, T. Recent advances in systems metabolic engineering tools and strategies.
Chen, G. A microbial polyhydroxyalkanoates PHA based bio- and materials industry. Chen, Y. Regulation of the adhE gene, which encodes ethanol dehydrogenase in Escherichia coli. Clark, D. The fermentation pathways of Escherichia coli. FEMS Microbiol. Diehl, A. Quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa is a homodimer - Sequence of the gene and deduced structural properties of the enzyme. Echave, P. DnaK dependence of mutant ethanol oxidoreductases evolved for aerobic function and protective role of the chaperone against protein oxidative damage in Escherichia coli.
Enjalbert, B. Acetate exposure determines the diauxic behavior of Escherichia coli during the glucose-acetate transition. Farmer, W. Reduction of aerobic acetate production by Escherichia coli. Fic, E. Fox, D. Isolation and characterization of homogeneous acetate kinase from Salmonella typhimurium and Escherichia coli. Google Scholar. Gorke, B. We showed that glucose and acetate can be co-substrates for E. We investigated whether this could also be the case for other glycolytic carbon sources, notably for some nutrients assumed to support growth of E.
This was tested by growing E. As observed on glucose, acetate was produced from gluconate or fucose as sole carbon sources. These observations extend the findings made on glucose and indicate that acetate is not only a by-product of glycolytic nutrients but can also be their co-substrate, depending solely on its extracellular concentration.
The data reported in this work show that E. This acetate consumption is not supported by acetyl-CoA synthetase. Not only is this enzyme repressed by catabolite repression in these conditions, but the simultaneous consumption of glucose and acetate is observed — to the same extent and even at very high acetate concentrations — in a mutant deleted for the acs gene.
The potential contribution of PoxB to this phenomenon can also be eliminated. This acetate utilization is supported by the Pta-AckA pathway. Though known to be reversible, the latter pathway is considered to be involved only in acetate production. Our data show that the Pta-AckA pathway alone can support both the production and consumption of acetate upon excess glucose.
Indeed, the pathway allows two opposite processes to occur concomitantly, including the conversion of acetyl-CoA into acetate and the reverse conversion of acetate into acetyl-CoA.
The two unidirectional fluxes were measured and were significant. They were in the range of glucose uptake, and were 3—4 times higher than the net flux of acetate production or utilization. The overall result acetate production or consumption is the net balance between the two opposite processes.
The net direction of the Pta-AckA pathway upon excess glucose, i. Above this threshold value, acetate is consumed. The two main pathways of acetate metabolism in E. Acs is controlled by catabolite repression, which is an active regulation process. This repression prevents the operation of the Pta-AckA-Acs cycle and hence avoids the spillage of energy.
The Pta-AckA pathway is controlled thermodynamically — i. First, the unidirectional fluxes of acetate production and acetate utilization are instantaneously adapted to the external concentration of acetate.
This allows fine tuning of acetate metabolism according to its availability. Moreover, this regulatory mechanism allows metabolism to switch rapidly from acetate dissimilation to acetate assimilation during glycolytic growth, for instance in response to a sudden increase in acetate availability.
This ability to react fast is likely beneficial to face sudden changes in acetate concentration and might represent a competitive advantage in particular environments or conditions. This illustrates that metabolism is not self-contained in terms of control, but is highly sensitive to the environment. Finally, acetate assimilation via Acs can be seen as an effective but expensive way — due to the operation of the Pta-AckA-Acs cycle — to assimilate acetate in low or limited carbon environments whereas the Pta-AckA pathway is a low cost process to scavenge acetate in C-reach environments.
Acetyl-phosphate, the intermediate of the Pta-AckA pathway, is known to regulate many cellular processes in E. It is tempting to speculate that, due to the thermodynamic control of the Pta-AckA pathway, the intracellular level of acetyl-phosphate, hence its regulatory role, is modulated according to acetate availability.
Indeed, our kinetic model predicts the accumulation of acetyl-phosphate when the extracellular concentration of acetate increases. These predicted concentrations are in good agreement with the intracellular levels of acetyl-phosphate reported in the literature 22 , 24 , 25 , The amplitude of the variation in acetyl-phosphate concentrations due to the thermodynamic control of the Pta-AckA pathway, as given by the model, is about 3 orders of magnitude.
This is significant and consistent with the role of acetyl-phosphate as regulator, in particular in increasing the acetylation level of proteins in vivo Hence, the extracellular concentration of acetate might modulate the acetyl-phosphate pool through the thermodynamic control of the Pta-AckA pathway. This, in turn, would regulate many cellular processes, and likely result in fine tuning of metabolism according to acetate availability.
Our data indicate that acetate metabolism in E. This control allows the bacterium to consume acetate even in conditions of glucose excess, provided the external concentration in acetate is high enough. This contrasts with the general consideration that acetate can be consumed only after exhaustion of glycolytic carbon sources. Our results indicate that the mode of utilization of glycolytic substrates and acetate by E. Neither glucose uptake nor cell growth are altered in this condition, indicating that net acetate production is not an absolute requirement for E.
In the laboratory, growth of E. In such conditions, the thermodynamics of the Pta-AckA pathway favors acetate production. The net production of acetate upon exponential growth on glucose can be the result of the thermodynamic driving force in the Pta-AckA pathway that drives acetate metabolism towards acetate production.
From the thermodynamic point of view, such levels of acetate favor the net consumption of this compound in the intestine. It is therefore very likely that E. Escherichia coli K MG was selected as the model wild-type strain. BW mutants 33 were used to create their equivalent in the MG background by bacteriophage P1-mediated transduction. The list of strains is given in Supplementary Table S2.
Sodium acetate prepared in solution at pH 7 was added to reach the required concentration. Acetate labelling assay was performed by adding 1. For 13 C-labeling experiments designed for flux calculation, unlabeled glucose was replaced by uniformly 13 C-labeled glucose Eurisotop, France. Concentrations of labeled and unlabeled glucose and acetate were quantified in filtered broth 0.
Intracellular metabolites were extracted from cells exponentially-growing on glucose without acetate initially present in the medium using the differential sampling method. The 13 C contents of intracellular metabolites were quantified from their isotopic patterns after correction for naturally abundant isotopes using the IsoCor software To quantify acetate production and consumption fluxes, a dynamic model describing the propagation of 13 C-atoms through the metabolic network was developed.
This model includes seven reactions that represent glucose uptake, glycolysis, acetate production and consumption, and growth.
Note that the system boundary considered here is the shake flask and not the cell. The dynamics of seven variables concentrations of biomass and of labeled and unlabeled glucose, acetate and AcCoA were simulated using the following system of ordinary differential equations ODEs :. The system of ODEs defined by Eqs 1—7 was implemented in Fortran , and simulations were performed using the lsoda function of the deSolve package of R v2.
Sensitivity analyses were carried out using the Monte-Carlo method with 1, iterations. The scripts used to perform 13 C-flux calculations are distributed in Supplementary Information under an open source license to ensure reproducibility and reusability. To investigate the impact of changes of acetate concentration on the flux through the Pta-AckA pathway, a kinetic model of this pathway was constructed.
The R scripts used to perform these simulations and generate the figures are provided in Supplementary Information. How to cite this article : Enjalbert, B.
Acetate fluxes in Escherichia coli are determined by the thermodynamic control of the Pta-AckA pathway. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Harden, A. The chemical action of Bacillus coli communis and similar organisms on carbohydrates and allied compounds. CAS Google Scholar. Cell Factories 8 , 54 Google Scholar. Chong, H. PloS One 8 , e Jian, J. Production of polyhydroxyalkanoates by Escherichia coli mutants with defected mixed acid fermentation pathways.
Luli, G. Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations. Oxygen availability and the growth of Escherichia coli. Bioprocess Eng. Wolfe, A. The acetate switch.
MMBR 69 , 12—50 Abdel-Hamid, A. Pyruvate oxidase contributes to the aerobic growth efficiency of Escherichia coli. Chang, Y. Expression of Escherichia coli pyruvate oxidase PoxB depends on the sigma factor encoded by the rpoS katF gene. Kakuda, H. Identification and characterization of the ackA acetate kinase A -pta phosphotransacetylase operon and complementation analysis of acetate utilization by an ackA-pta deletion mutant of Escherichia coli. Tokyo , — Kumari, S.
Regulation of acetyl coenzyme A synthetase in Escherichia coli. Cloning, characterization, and functional expression of acs, the gene which encodes acetyl coenzyme A synthetase in Escherichia coli. Flores, N. Adaptation for fast growth on glucose by differential expression of central carbon metabolism and gal regulon genes in an Escherichia coli strain lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system.
PubMed Google Scholar. Valgepea, K. Decrease of energy spilling in Escherichia coli continuous cultures with rising specific growth rate and carbon wasting. BMC Syst. Renilla, S. Acetate scavenging activity in Escherichia coli : interplay of acetyl—CoA synthetase and the PEP—glyoxylate cycle in chemostat cultures. Systems biology approach reveals that overflow metabolism of acetate in Escherichia coli is triggered by carbon catabolite repression of acetyl-CoA synthetase.
Kotte, O. Orth, J. A comprehensive genome-scale reconstruction of Escherichia coli metabolism— Keywords: dehydrogenase; flux analysis; glycolysis; lipogenesis; metabolomics; mitochondria; pyruvate; reactive oxygen species; stable isotope tracing; thiamine. Abstract Acetate is a major nutrient that supports acetyl-coenzyme A Ac-CoA metabolism and thus lipogenesis and protein acetylation.
Publication types Research Support, N.
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