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High-flux isobutanol production using engineered Escherichia coli: a bioreactor study with in situ product removal.

Baez A, Cho KM, Liao JC - Appl. Microbiol. Biotechnol. (2011)

Bottom Line: Promising approaches to produce higher alcohols, e.g., isobutanol, using Escherichia coli have been developed with successful results.Here, we translated the isobutanol process from shake flasks to a 1-L bioreactor in order to characterize three E. coli strains.The isobutanol productivity was approximately twofold and the titer was 9% higher than n-butanol produced by Clostridium in a similar integrated system.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, University of California-Los Angeles, 420 Westwood Plaza, Los Angeles, CA 90095, USA.

ABSTRACT
Promising approaches to produce higher alcohols, e.g., isobutanol, using Escherichia coli have been developed with successful results. Here, we translated the isobutanol process from shake flasks to a 1-L bioreactor in order to characterize three E. coli strains. With in situ isobutanol removal from the bioreactor using gas stripping, the engineered E. coli strain (JCL260) produced more than 50 g/L in 72 h. In addition, the isobutanol production by the parental strain (JCL16) and the high isobutanol-tolerant mutant (SA481) were compared with JCL260. Interestingly, we found that the isobutanol-tolerant strain in fact produced worse than either JCL16 or JCL260. This result suggests that in situ product removal can properly overcome isobutanol toxicity in E. coli cultures. The isobutanol productivity was approximately twofold and the titer was 9% higher than n-butanol produced by Clostridium in a similar integrated system.

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Enzyme activity of isobutanol pathway for JCL260 carrying pSA65/pSA69 plasmids in bioreactor cultures at 30°C (open symbols) and 37°C (closed symbols). a Total isobutanol production calculated as sum of isobutanol concentrations from receivers B, D (Fig. 1), and broth culture considering a working volume of 0.35 L. b Time profile of glucose concentration and cell growth. c Acetolactate synthase (AlsS) activity. d Dihydroxy-acid dehydratase activity. e 2-Ketoisovalerate decarboxylase (KivD) activity. f Alcohol dehydrogenase (AdhA) activity. Error bars indicate the difference between duplicate assays
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Fig6: Enzyme activity of isobutanol pathway for JCL260 carrying pSA65/pSA69 plasmids in bioreactor cultures at 30°C (open symbols) and 37°C (closed symbols). a Total isobutanol production calculated as sum of isobutanol concentrations from receivers B, D (Fig. 1), and broth culture considering a working volume of 0.35 L. b Time profile of glucose concentration and cell growth. c Acetolactate synthase (AlsS) activity. d Dihydroxy-acid dehydratase activity. e 2-Ketoisovalerate decarboxylase (KivD) activity. f Alcohol dehydrogenase (AdhA) activity. Error bars indicate the difference between duplicate assays

Mentions: Since vapor pressure increases with temperature, higher temperature should increase the gas stripping rate, and consequently, isobutanol concentration in the fermentation broth should be lower. Accordingly, the isobutanol production by JCL260 was evaluated at 37°C and compared with the production at 30°C (Fig. 2a). Unfortunately, the maximum isobutanol concentration was twofold lower at 37°C compared with that at 30°C. The isobutanol concentration in the fermentation broth reached 5.3 g/L and decreased until 0.4 g/L at the end of culture (Fig. 2b). The maximum biomass concentration (7.2 g/L) was practically the same for both temperatures (Fig. 2c). Glucose depletion was avoided by intermittent feeding in all cultures (Fig. 2d). Acetate production was higher at 37°C (Fig. 2e), reaching a maximum concentration of 15.8 ± 1.6 g/L at 10.5 h. Initially, it was hypothesized that the lower isobutanol production at 37°C was due to the poor quality of heterologous protein expression. To test this hypothesis, activities of four (AlsS, IlvD, KivD, and AdhA) enzymes involved in the biosynthetic isobutanol pathway were determined in bioreactor cultures performed at 30°C and 37°C (Fig. 6). Isobutanol, growth, and glucose time profile are shown in Fig. 6a, b, and in concordance with the results of Fig. 2a, maximum isobutanol concentration reached at 30°C was twofold higher than at 37°C. Enzyme activities of AlsS, IlvD, and KivD were roughly similar in both 30°C and 37°C cultures (Fig. 6c–e). However, the AdhA activity (Fig. 6f) in the 37°C culture was significantly lower than that in the 30°C culture from 17 h to the end of cultures, suggesting that this step may contribute to the difference between the two temperatures.Fig. 6


High-flux isobutanol production using engineered Escherichia coli: a bioreactor study with in situ product removal.

Baez A, Cho KM, Liao JC - Appl. Microbiol. Biotechnol. (2011)

Enzyme activity of isobutanol pathway for JCL260 carrying pSA65/pSA69 plasmids in bioreactor cultures at 30°C (open symbols) and 37°C (closed symbols). a Total isobutanol production calculated as sum of isobutanol concentrations from receivers B, D (Fig. 1), and broth culture considering a working volume of 0.35 L. b Time profile of glucose concentration and cell growth. c Acetolactate synthase (AlsS) activity. d Dihydroxy-acid dehydratase activity. e 2-Ketoisovalerate decarboxylase (KivD) activity. f Alcohol dehydrogenase (AdhA) activity. Error bars indicate the difference between duplicate assays
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3094657&req=5

Fig6: Enzyme activity of isobutanol pathway for JCL260 carrying pSA65/pSA69 plasmids in bioreactor cultures at 30°C (open symbols) and 37°C (closed symbols). a Total isobutanol production calculated as sum of isobutanol concentrations from receivers B, D (Fig. 1), and broth culture considering a working volume of 0.35 L. b Time profile of glucose concentration and cell growth. c Acetolactate synthase (AlsS) activity. d Dihydroxy-acid dehydratase activity. e 2-Ketoisovalerate decarboxylase (KivD) activity. f Alcohol dehydrogenase (AdhA) activity. Error bars indicate the difference between duplicate assays
Mentions: Since vapor pressure increases with temperature, higher temperature should increase the gas stripping rate, and consequently, isobutanol concentration in the fermentation broth should be lower. Accordingly, the isobutanol production by JCL260 was evaluated at 37°C and compared with the production at 30°C (Fig. 2a). Unfortunately, the maximum isobutanol concentration was twofold lower at 37°C compared with that at 30°C. The isobutanol concentration in the fermentation broth reached 5.3 g/L and decreased until 0.4 g/L at the end of culture (Fig. 2b). The maximum biomass concentration (7.2 g/L) was practically the same for both temperatures (Fig. 2c). Glucose depletion was avoided by intermittent feeding in all cultures (Fig. 2d). Acetate production was higher at 37°C (Fig. 2e), reaching a maximum concentration of 15.8 ± 1.6 g/L at 10.5 h. Initially, it was hypothesized that the lower isobutanol production at 37°C was due to the poor quality of heterologous protein expression. To test this hypothesis, activities of four (AlsS, IlvD, KivD, and AdhA) enzymes involved in the biosynthetic isobutanol pathway were determined in bioreactor cultures performed at 30°C and 37°C (Fig. 6). Isobutanol, growth, and glucose time profile are shown in Fig. 6a, b, and in concordance with the results of Fig. 2a, maximum isobutanol concentration reached at 30°C was twofold higher than at 37°C. Enzyme activities of AlsS, IlvD, and KivD were roughly similar in both 30°C and 37°C cultures (Fig. 6c–e). However, the AdhA activity (Fig. 6f) in the 37°C culture was significantly lower than that in the 30°C culture from 17 h to the end of cultures, suggesting that this step may contribute to the difference between the two temperatures.Fig. 6

Bottom Line: Promising approaches to produce higher alcohols, e.g., isobutanol, using Escherichia coli have been developed with successful results.Here, we translated the isobutanol process from shake flasks to a 1-L bioreactor in order to characterize three E. coli strains.The isobutanol productivity was approximately twofold and the titer was 9% higher than n-butanol produced by Clostridium in a similar integrated system.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, University of California-Los Angeles, 420 Westwood Plaza, Los Angeles, CA 90095, USA.

ABSTRACT
Promising approaches to produce higher alcohols, e.g., isobutanol, using Escherichia coli have been developed with successful results. Here, we translated the isobutanol process from shake flasks to a 1-L bioreactor in order to characterize three E. coli strains. With in situ isobutanol removal from the bioreactor using gas stripping, the engineered E. coli strain (JCL260) produced more than 50 g/L in 72 h. In addition, the isobutanol production by the parental strain (JCL16) and the high isobutanol-tolerant mutant (SA481) were compared with JCL260. Interestingly, we found that the isobutanol-tolerant strain in fact produced worse than either JCL16 or JCL260. This result suggests that in situ product removal can properly overcome isobutanol toxicity in E. coli cultures. The isobutanol productivity was approximately twofold and the titer was 9% higher than n-butanol produced by Clostridium in a similar integrated system.

Show MeSH
Related in: MedlinePlus