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Model-driven intracellular redox status modulation for increasing isobutanol production in Escherichia coli.

Liu J, Qi H, Wang C, Wen J - Biotechnol Biofuels (2015)

Bottom Line: Few strains have been found to produce isobutanol naturally.The redox modulations resulted in the decrease production of ethanol and lactate by 17.5 and 51.7% to 1.32 and 6.08 g/L, respectively.Moreover, the developed model-driven method special for redox cofactor metabolism was of very helpful to the redox status modulation of other bio-products.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, 300072 People's Republic of China ; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072 People's Republic of China.

ABSTRACT

Background: Few strains have been found to produce isobutanol naturally. For building a high performance isobutanol-producing strain, rebalancing redox status of the cell was very crucial through systematic investigation of redox cofactors metabolism. Then, the metabolic model provided a powerful tool for the rational modulation of the redox status.

Results: Firstly, a starting isobutanol-producing E. coli strain LA02 was engineered with only 2.7 g/L isobutanol produced. Then, the genome-scale metabolic modeling was specially carried out for the redox cofactor metabolism of the strain LA02 by combining flux balance analysis and minimization of metabolic adjustment, and the GAPD reaction catalyzed by the glyceraldehyde-3-phosphate dehydrogenase was predicted as the key target for redox status improvement. Under guidance of the metabolic model prediction, a gapN-encoding NADP(+) dependent glyceraldehyde-3-phosphate dehydrogenase pathway was constructed and then fine-tuned using five constitutive promoters. The best strain LA09 was obtained with the strongest promoter BBa_J23100. The NADPH/NADP + ratios of strain LA09 reached 0.67 at exponential phase and 0.64 at stationary phase. The redox modulations resulted in the decrease production of ethanol and lactate by 17.5 and 51.7% to 1.32 and 6.08 g/L, respectively. Therefore, the isobutanol titer was increased by 221% to 8.68 g/L.

Conclusions: This research has achieved rational redox status improvement of isobutanol-producing strain under guidance of the prediction and modeling of the genome-scale metabolic model of isobutanol-producing E. coli strain with the aid of synthetic promoters. Therefore, the production of isobutanol was dramatically increased by 2.21-fold from 2.7 to 8.68 g/L. Moreover, the developed model-driven method special for redox cofactor metabolism was of very helpful to the redox status modulation of other bio-products.

No MeSH data available.


Related in: MedlinePlus

Simulation of metabolic flux changes in the central metabolism of the redox reaction manipulated strains. In each case, all fluxes were expressed in molar percentage of the corresponding glucose uptake rate. The red lines indicated reactions with NADP(H) as cofactor, the thin blue lines indicated reactions with NAD(H) as cofactor, the green lines indicated ATP generating/consuming reactions, the thick blue lines indicated the redox synthesis pathway of succinate, the dot lines indicated the transportation reactions from intracellular to extracellular. 1,3DPG 3-phospho-glyceroyl phosphate, 2PG glycerate 2-phosphate, 3PG 3-phospho-glycerate, 6PGC 6-phospho-gluconate, 6PGL 6-phospho-glucono-1,5-lactone, ACCOA acetyl-CoA, ALAC 2-acetolactate, AKG 2-oxoglutarate, CIT citrate, DHKIV 2,3-dihydroxy-3-methylbutanoate, E4P erythrose 4-phosphate, F6P fructose 6-phosphate, FDP fructose-1,6-diphosphate, FUM fumarate, G3P glyceraldehyde 3-phosphate, G6P glucose 6-phosphate, GLC glucose, IBTA isobutaldehyde, ICIT isocitrate, KIV α-ketoisovaleric acid, l-MALl-malate, OAA oxaloacetate, PEP phosphoenolpyruvate, PYR pyruvate, R5P ribose 5-phosphate, RL5P ribulose 5-phosphate, S7P sedoheptulose 7-phosphate, SUCCOA succiny-CoA, X5P xylulose 5-phosphate.
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Fig1: Simulation of metabolic flux changes in the central metabolism of the redox reaction manipulated strains. In each case, all fluxes were expressed in molar percentage of the corresponding glucose uptake rate. The red lines indicated reactions with NADP(H) as cofactor, the thin blue lines indicated reactions with NAD(H) as cofactor, the green lines indicated ATP generating/consuming reactions, the thick blue lines indicated the redox synthesis pathway of succinate, the dot lines indicated the transportation reactions from intracellular to extracellular. 1,3DPG 3-phospho-glyceroyl phosphate, 2PG glycerate 2-phosphate, 3PG 3-phospho-glycerate, 6PGC 6-phospho-gluconate, 6PGL 6-phospho-glucono-1,5-lactone, ACCOA acetyl-CoA, ALAC 2-acetolactate, AKG 2-oxoglutarate, CIT citrate, DHKIV 2,3-dihydroxy-3-methylbutanoate, E4P erythrose 4-phosphate, F6P fructose 6-phosphate, FDP fructose-1,6-diphosphate, FUM fumarate, G3P glyceraldehyde 3-phosphate, G6P glucose 6-phosphate, GLC glucose, IBTA isobutaldehyde, ICIT isocitrate, KIV α-ketoisovaleric acid, l-MALl-malate, OAA oxaloacetate, PEP phosphoenolpyruvate, PYR pyruvate, R5P ribose 5-phosphate, RL5P ribulose 5-phosphate, S7P sedoheptulose 7-phosphate, SUCCOA succiny-CoA, X5P xylulose 5-phosphate.

Mentions: In the simulation, the specific isobutanol production rate could be increased only by ten potential targets manipulation (Table 2), while the modeling results of the other potential targets are presented in the Additional file 1: Table S6. With the highest value of fPH (1.944), the reaction GAPDH was identified as the most important target. Similarly, the GAPDH was also predicted by the reference [11] to be the best cofactor-swap target in E. coli for enhancing the theoretical yields of several native and non-native products. The cofactor-swap of GAPDH could not only regulate the cofactor metabolism but also modulate itself carbon flux to impact the cell growth and isobutanol production. On the one hand, the limited NADPH and excess NADH were generated in E. coli MG1655 [25–28], which could be overcome with the new efficient NADPH-generating reaction obtained by cofactor-swap of GAPDH. On the other hand, the reaction GAPDH was a key step of the EMP pathway, which dominated the glucose catabolism, and its manipulation could increase the availability of pyruvate as the end product of the EMP pathway. The predicted specific isobutanol production was increased by 183.0%, while the specific production rate of the byproducts, ethanol and lactate, was decreased by 20.1 and 14.8%, respectively (Table 2; Fig. 1).Table 2


Model-driven intracellular redox status modulation for increasing isobutanol production in Escherichia coli.

Liu J, Qi H, Wang C, Wen J - Biotechnol Biofuels (2015)

Simulation of metabolic flux changes in the central metabolism of the redox reaction manipulated strains. In each case, all fluxes were expressed in molar percentage of the corresponding glucose uptake rate. The red lines indicated reactions with NADP(H) as cofactor, the thin blue lines indicated reactions with NAD(H) as cofactor, the green lines indicated ATP generating/consuming reactions, the thick blue lines indicated the redox synthesis pathway of succinate, the dot lines indicated the transportation reactions from intracellular to extracellular. 1,3DPG 3-phospho-glyceroyl phosphate, 2PG glycerate 2-phosphate, 3PG 3-phospho-glycerate, 6PGC 6-phospho-gluconate, 6PGL 6-phospho-glucono-1,5-lactone, ACCOA acetyl-CoA, ALAC 2-acetolactate, AKG 2-oxoglutarate, CIT citrate, DHKIV 2,3-dihydroxy-3-methylbutanoate, E4P erythrose 4-phosphate, F6P fructose 6-phosphate, FDP fructose-1,6-diphosphate, FUM fumarate, G3P glyceraldehyde 3-phosphate, G6P glucose 6-phosphate, GLC glucose, IBTA isobutaldehyde, ICIT isocitrate, KIV α-ketoisovaleric acid, l-MALl-malate, OAA oxaloacetate, PEP phosphoenolpyruvate, PYR pyruvate, R5P ribose 5-phosphate, RL5P ribulose 5-phosphate, S7P sedoheptulose 7-phosphate, SUCCOA succiny-CoA, X5P xylulose 5-phosphate.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4522091&req=5

Fig1: Simulation of metabolic flux changes in the central metabolism of the redox reaction manipulated strains. In each case, all fluxes were expressed in molar percentage of the corresponding glucose uptake rate. The red lines indicated reactions with NADP(H) as cofactor, the thin blue lines indicated reactions with NAD(H) as cofactor, the green lines indicated ATP generating/consuming reactions, the thick blue lines indicated the redox synthesis pathway of succinate, the dot lines indicated the transportation reactions from intracellular to extracellular. 1,3DPG 3-phospho-glyceroyl phosphate, 2PG glycerate 2-phosphate, 3PG 3-phospho-glycerate, 6PGC 6-phospho-gluconate, 6PGL 6-phospho-glucono-1,5-lactone, ACCOA acetyl-CoA, ALAC 2-acetolactate, AKG 2-oxoglutarate, CIT citrate, DHKIV 2,3-dihydroxy-3-methylbutanoate, E4P erythrose 4-phosphate, F6P fructose 6-phosphate, FDP fructose-1,6-diphosphate, FUM fumarate, G3P glyceraldehyde 3-phosphate, G6P glucose 6-phosphate, GLC glucose, IBTA isobutaldehyde, ICIT isocitrate, KIV α-ketoisovaleric acid, l-MALl-malate, OAA oxaloacetate, PEP phosphoenolpyruvate, PYR pyruvate, R5P ribose 5-phosphate, RL5P ribulose 5-phosphate, S7P sedoheptulose 7-phosphate, SUCCOA succiny-CoA, X5P xylulose 5-phosphate.
Mentions: In the simulation, the specific isobutanol production rate could be increased only by ten potential targets manipulation (Table 2), while the modeling results of the other potential targets are presented in the Additional file 1: Table S6. With the highest value of fPH (1.944), the reaction GAPDH was identified as the most important target. Similarly, the GAPDH was also predicted by the reference [11] to be the best cofactor-swap target in E. coli for enhancing the theoretical yields of several native and non-native products. The cofactor-swap of GAPDH could not only regulate the cofactor metabolism but also modulate itself carbon flux to impact the cell growth and isobutanol production. On the one hand, the limited NADPH and excess NADH were generated in E. coli MG1655 [25–28], which could be overcome with the new efficient NADPH-generating reaction obtained by cofactor-swap of GAPDH. On the other hand, the reaction GAPDH was a key step of the EMP pathway, which dominated the glucose catabolism, and its manipulation could increase the availability of pyruvate as the end product of the EMP pathway. The predicted specific isobutanol production was increased by 183.0%, while the specific production rate of the byproducts, ethanol and lactate, was decreased by 20.1 and 14.8%, respectively (Table 2; Fig. 1).Table 2

Bottom Line: Few strains have been found to produce isobutanol naturally.The redox modulations resulted in the decrease production of ethanol and lactate by 17.5 and 51.7% to 1.32 and 6.08 g/L, respectively.Moreover, the developed model-driven method special for redox cofactor metabolism was of very helpful to the redox status modulation of other bio-products.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, 300072 People's Republic of China ; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072 People's Republic of China.

ABSTRACT

Background: Few strains have been found to produce isobutanol naturally. For building a high performance isobutanol-producing strain, rebalancing redox status of the cell was very crucial through systematic investigation of redox cofactors metabolism. Then, the metabolic model provided a powerful tool for the rational modulation of the redox status.

Results: Firstly, a starting isobutanol-producing E. coli strain LA02 was engineered with only 2.7 g/L isobutanol produced. Then, the genome-scale metabolic modeling was specially carried out for the redox cofactor metabolism of the strain LA02 by combining flux balance analysis and minimization of metabolic adjustment, and the GAPD reaction catalyzed by the glyceraldehyde-3-phosphate dehydrogenase was predicted as the key target for redox status improvement. Under guidance of the metabolic model prediction, a gapN-encoding NADP(+) dependent glyceraldehyde-3-phosphate dehydrogenase pathway was constructed and then fine-tuned using five constitutive promoters. The best strain LA09 was obtained with the strongest promoter BBa_J23100. The NADPH/NADP + ratios of strain LA09 reached 0.67 at exponential phase and 0.64 at stationary phase. The redox modulations resulted in the decrease production of ethanol and lactate by 17.5 and 51.7% to 1.32 and 6.08 g/L, respectively. Therefore, the isobutanol titer was increased by 221% to 8.68 g/L.

Conclusions: This research has achieved rational redox status improvement of isobutanol-producing strain under guidance of the prediction and modeling of the genome-scale metabolic model of isobutanol-producing E. coli strain with the aid of synthetic promoters. Therefore, the production of isobutanol was dramatically increased by 2.21-fold from 2.7 to 8.68 g/L. Moreover, the developed model-driven method special for redox cofactor metabolism was of very helpful to the redox status modulation of other bio-products.

No MeSH data available.


Related in: MedlinePlus