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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 intracellular redox cofactors in exponential phase (a), isobutanol and biomass (b) for strain LA02, LA03 and LA04. Asterisks indicate significant differences (*P < 0.05, **P < 0.01).
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Fig2: Comparison of intracellular redox cofactors in exponential phase (a), isobutanol and biomass (b) for strain LA02, LA03 and LA04. Asterisks indicate significant differences (*P < 0.05, **P < 0.01).

Mentions: The two genes gapC and gapN were cloned and expressed in strain LA02, respectively, resulting in strains LA03 and LA04 (see Table 1). Then, the intercellular redox cofactors pool at exponential phase (EP) (18 h) and stationary phase (SP) (36 h) of bath culture were determined. Since similar results and conclusions were drawn from the data of EP and SP, the results at EP are presented in Fig. 2a and the results at SP are given in the Additional file 1: Figure S2. Previous reports had shown that the NADH/NAD+ ratio of the wild-type strain E. coli MG1655 ranged from 0.375 to 0.65, while the NADPH/NADP+ ratio varied from 0.65 to 0.99 [27, 28, 37]. In this work, the isobutanol was produced by the strain LA02 with lots of NADPH consumed, thus the NADPH/NADP+ ratio of the strain LA02 was decreased to 0.35 (EP) lower than that of the wild-type strain MG1655, while the NADH was oversupplied with a high NADH/NAD+ ratio of 0.90 (EP). Obviously, the redox status of strain LA02 was imbalance. As shown in Fig. 2, the redox status of strain LA03 was very close to that of strain LA02, which indicated that little improvement of the redox status had been made by gapC expression. Consequently, the isobutanol produced by strain LA03 was only 3.10 g/L, a slight increase compared to that of LA02 (2.70 g/L), and the isobutanol yield of LA03 (0.238 mol/mol, 28.3% of theoretical yield) was very similar to that of LA02 (0.240 mol/mol, 28.6% of theoretical yield). Unlike gapC expression, the gapN expression obviously made an increase of NADPH/NAPD+ ratio to 0.56 (EP) in strain LA04, 1.6-fold higher than that of strain LA02 (P < 0.01). Moreover, the GAPDN expression could reduce the NADH generation via the native GAPDH by competing for the same substrate of glyceraldehyde 3-phosphate (G3P). Thus, in strain LA04, the NADH/NAD+ ratio was decreased by 18.9% (EP) compared to that in strain LA02 (P < 0.01) (Fig. 2a). These results indicated the redox status in strain LA04 was improved by the gapN expression, and therefore the isobutanol production (5.28 g/L) and yield (0.38 mol/mol, 45.2% of theoretical yield) were both increased by 95.6 and 58.3% compared to those of strain LA02 (P < 0.01). Additionally, the final biomass of LA04 was also increased to 2.08 g/L dry cell weight (DCW), 6.7% higher than that of LA02 (P < 0.05) (Fig. 2c). No ATP release by the non-phosphorylating reaction catalyzed by GapN has been taken into account. The less ATP production would result in slow growth [38] and higher glucose uptake rate [39]. However, NADPH also played an important role in the biosynthesis of fatty acid, protein and nucleic acid, and was also a key metabolite to resist oxidative stress [40]. Rebalancing redox status with an increase of NADPH level would help the cell to resist the oxidative stress caused by isobutanol [8, 41, 42].Fig. 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)

Comparison of intracellular redox cofactors in exponential phase (a), isobutanol and biomass (b) for strain LA02, LA03 and LA04. 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

Fig2: Comparison of intracellular redox cofactors in exponential phase (a), isobutanol and biomass (b) for strain LA02, LA03 and LA04. Asterisks indicate significant differences (*P < 0.05, **P < 0.01).
Mentions: The two genes gapC and gapN were cloned and expressed in strain LA02, respectively, resulting in strains LA03 and LA04 (see Table 1). Then, the intercellular redox cofactors pool at exponential phase (EP) (18 h) and stationary phase (SP) (36 h) of bath culture were determined. Since similar results and conclusions were drawn from the data of EP and SP, the results at EP are presented in Fig. 2a and the results at SP are given in the Additional file 1: Figure S2. Previous reports had shown that the NADH/NAD+ ratio of the wild-type strain E. coli MG1655 ranged from 0.375 to 0.65, while the NADPH/NADP+ ratio varied from 0.65 to 0.99 [27, 28, 37]. In this work, the isobutanol was produced by the strain LA02 with lots of NADPH consumed, thus the NADPH/NADP+ ratio of the strain LA02 was decreased to 0.35 (EP) lower than that of the wild-type strain MG1655, while the NADH was oversupplied with a high NADH/NAD+ ratio of 0.90 (EP). Obviously, the redox status of strain LA02 was imbalance. As shown in Fig. 2, the redox status of strain LA03 was very close to that of strain LA02, which indicated that little improvement of the redox status had been made by gapC expression. Consequently, the isobutanol produced by strain LA03 was only 3.10 g/L, a slight increase compared to that of LA02 (2.70 g/L), and the isobutanol yield of LA03 (0.238 mol/mol, 28.3% of theoretical yield) was very similar to that of LA02 (0.240 mol/mol, 28.6% of theoretical yield). Unlike gapC expression, the gapN expression obviously made an increase of NADPH/NAPD+ ratio to 0.56 (EP) in strain LA04, 1.6-fold higher than that of strain LA02 (P < 0.01). Moreover, the GAPDN expression could reduce the NADH generation via the native GAPDH by competing for the same substrate of glyceraldehyde 3-phosphate (G3P). Thus, in strain LA04, the NADH/NAD+ ratio was decreased by 18.9% (EP) compared to that in strain LA02 (P < 0.01) (Fig. 2a). These results indicated the redox status in strain LA04 was improved by the gapN expression, and therefore the isobutanol production (5.28 g/L) and yield (0.38 mol/mol, 45.2% of theoretical yield) were both increased by 95.6 and 58.3% compared to those of strain LA02 (P < 0.01). Additionally, the final biomass of LA04 was also increased to 2.08 g/L dry cell weight (DCW), 6.7% higher than that of LA02 (P < 0.05) (Fig. 2c). No ATP release by the non-phosphorylating reaction catalyzed by GapN has been taken into account. The less ATP production would result in slow growth [38] and higher glucose uptake rate [39]. However, NADPH also played an important role in the biosynthesis of fatty acid, protein and nucleic acid, and was also a key metabolite to resist oxidative stress [40]. Rebalancing redox status with an increase of NADPH level would help the cell to resist the oxidative stress caused by isobutanol [8, 41, 42].Fig. 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