Improving sustainable isopropanol production in engineered Escherichia coli W via oxygen limitation

Abstract

Background: Due to ecological concerns, alternative supply lines for fuel and bulk chemicals such as isopropanol are increasingly pursued. By implementing the formation pathways from natural producers like Clostridium beijerinckii and Clostridium aurantibutyricum, isopropanol can be produced in Escherichia coli. However, developing an industrially and economically feasible microbial production process requires a robust and efficient process strategy. Therefore, this study explores microaerobic conditions in combination with lactose and sour whey as sustainable carbon source as a basis for large-scale microbial isopropanol production.

Results: Different gas-liquid mass transfer regimes (affected by variations of the stirrer speed and ingas oxygen concentration) allowed the implementation of different microaerobic conditions characterized by their specific oxygen uptake rate (q_O2) in cultivations with an isopropanol producing E. coli W strain on lactose. Under microaerobic conditions, the specific isopropanol production rate (q_{p, ipa}) exhibited a direct correlation with q_O2. Moreover, isopropanol production showed a pseudo growth-coupled behavior. Monitoring of the formation rates of various by-products such as acetone, lactate, acetate, pyruvate, formate and succinate allowed to identify a q_O2 of 9.6 mmol g^{- 1} h^{- 1} in only slightly microaerobic cultivations as the best conditions for microbial isopropanol production. Additionally, the data suggests that a carbon bottleneck exists at the pyruvate node and the availability of the redox factor NADPH is crucial to shift the product balance from acetone to isopropanol. Finally, confirmation runs prove the effectiveness of the microaerobic production approach by yielding 8.2 g L^{- 1} (135.8 ± 13.3 mmol L^{- 1}) and 20.6 g L^{- 1} (342.9 ± 0.4 mmol L^{- 1}) isopropanol on lactose and whey, respectively, reaching a volumetric isopropanol formation rate of up to 2.44 g L^{- 1} h^{- 1} (40.6 mmol L^{- 1} h^{- 1}).

Conclusions: This study identifies slightly microaerobic conditions (q_O2 ~ 10 mmol g^{- 1} h^{- 1}) as the currently best conditions for microbial isopropanol production on lactose and whey in E. coli W. In the future, optimizing the carbon flux around the pyruvate node, ensuring sufficient NADPH supply, and establishing a feedback control loop to control process variables affecting oxygen transfer to the culture, could make microbial isopropanol production feasible at an industrial scale.

Keywords: Escherichia coli; Isopropanol; Lactose; Microaerobic conditions; Oxygen limitation; Sustainable bioprocessing; Whey.

Fig. 1

Metabolic network around the pyruvate and acetyl-CoA nodes in E. coli W engineered for isopropanol production

Fig. 2

Shake flask experiments on lactose with isopropanol producing E. coli W, E. coli KO2 and E. coli KO4. Profiles of OD₆₀₀ (a), lactose (b), acetate (c), pyruvate (d), acetone (e) and isopropanol (f) are shown as means of independent biological triplicates

Fig. 3

Concentration profiles during aerobic cultivation. Biomass, lactose, galactose, lactate, acetone, and isopropanol concentrations of isopropanol producing E. coli W in batch, fed-batch mode on whey

Fig. 4

Specific parameters during microaerobic phases at the DoE setpoints. Oxygen uptake rates (q_O2) (a), specific isopropanol production rates (q_{p, ipa}) (b) and isopropanol yield per carbon of lactose (Y_ipa/s)(c)

Fig. 5

Influence of the specific oxygen uptake rate (q_O2) on the specific metabolite formation rates. Isopropanol formation rate q_{p, ipa} (a), specific acetone formation rate q_{p, aco}(b), specific lactate formation rate q_{p, lac} (c), specific acetate formation rate q_{p, ace} (d), specific pyruvate formation rate q_{p, pyr} (e), specific formate formation rate q_{p, for} (f), specific succinate formation rate q_{p, suc} (g), and specific growth rate µ (h) during microaerobic phases in the DoE experiments

Fig. 6

Confirmation runs at the best DoE conditions. Microaerobic conditions defined by 1000 rpm and 30% O₂ in the ingas stream during, a) cultivation on lactose, b) cultivation on whey