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 (qO₂) in cultivations with an isopropanol producing E. coli W strain on lactose. Under microaerobic conditions, the specific isopropanol production rate (qp, ipa) exhibited a direct correlation with qO₂. 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 qO₂ 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⁻¹h⁻¹ (40.6 mmol L⁻¹h⁻¹). Conclusions This study identifies slightly microaerobic conditions (qO2 ~ 10 mmol g⁻¹h⁻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.