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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

Comparison of the gapN expression (a), isobutanol and biomass (b), intracellular redox cofactors in exponential (c) and stationary (d) phase, for strain LA05, LA06, LA07, LA08 and LA09. Asterisks indicate significant differences (*P < 0.05, **P < 0.01).
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Fig3: Comparison of the gapN expression (a), isobutanol and biomass (b), intracellular redox cofactors in exponential (c) and stationary (d) phase, for strain LA05, LA06, LA07, LA08 and LA09. Asterisks indicate significant differences (*P < 0.05, **P < 0.01).

Mentions: Although it was impossible to define what the best redox status for isobutanol production was, a fine-tuning of gapN over-expression allowed to modulate NADPH and NADH generation by redistributing the fluxes between GAPDN and the native GAPDH competing for the same substrate of G3P [44]. Thus, the gapN gene expression was regulated under five different strength constitutive promoters in this work. These promoters BBa_J23105, BBa_J23106, BBa_J23118, BBa_J23102 and BBa_J23100 were selected from Registry of Standard Biological Parts, the promoters are listed in the order of the weakest to the strongest. The 2−∆∆Ct values [45] of the gapN expression in the five strain LA05, LA06, LA07, LA08 and LA09 measured by quantitative real-time reverse transcription PCR (RT-PCR) were 0.184, 0.218, 0.253, 0.373 and 0.479, respectively (Fig. 3a), indicating a fine-tuned expression of gapN was achieved and would be helpful for get a moderate redox status.Fig. 3


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

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

Comparison of the gapN expression (a), isobutanol and biomass (b), intracellular redox cofactors in exponential (c) and stationary (d) phase, for strain LA05, LA06, LA07, LA08 and LA09. Asterisks indicate significant differences (*P < 0.05, **P < 0.01).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Comparison of the gapN expression (a), isobutanol and biomass (b), intracellular redox cofactors in exponential (c) and stationary (d) phase, for strain LA05, LA06, LA07, LA08 and LA09. Asterisks indicate significant differences (*P < 0.05, **P < 0.01).
Mentions: Although it was impossible to define what the best redox status for isobutanol production was, a fine-tuning of gapN over-expression allowed to modulate NADPH and NADH generation by redistributing the fluxes between GAPDN and the native GAPDH competing for the same substrate of G3P [44]. Thus, the gapN gene expression was regulated under five different strength constitutive promoters in this work. These promoters BBa_J23105, BBa_J23106, BBa_J23118, BBa_J23102 and BBa_J23100 were selected from Registry of Standard Biological Parts, the promoters are listed in the order of the weakest to the strongest. The 2−∆∆Ct values [45] of the gapN expression in the five strain LA05, LA06, LA07, LA08 and LA09 measured by quantitative real-time reverse transcription PCR (RT-PCR) were 0.184, 0.218, 0.253, 0.373 and 0.479, respectively (Fig. 3a), indicating a fine-tuned expression of gapN was achieved and would be helpful for get a moderate redox status.Fig. 3

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