Smirnova, U. (2023). Energy-coupled isopropanol biosynthesis by engineered E. coli W from acetate [Diploma Thesis, Technische Universität Wien]. reposiTUm. https://doi.org/10.34726/hss.2023.112772
Acetate is a low-cost carbon source for biotechnological processes that can be obtained from ecological resources such as CO2 or lignocellulosic wastewater. However, its high toxicity and energetically unfavourable metabolism require efficient process strategies,one of which is ATP-coupled production which can improve energy availability and enhance cell growth and/or production.In this study, we developed the E. coli W IPA ptb buk (pbu) strain, which produces ATP-coupled isopropanol (IPA) from acetate using the novel IPA ptb buk pathway. This pathway employs the Ptb and Buk enzymes derived from C. acetobutylicum. The Ptbenzyme converts acetoacetyl-CoA to acetoacetylphosphate, which is then converted by the Buk enzyme to acetoacetate while generating one ATP molecule. According to the literature, the conversion of acetoacetyl-CoA to acetoacetate was typically accomplished in a single step using the AtoDA enzyme. Nonetheless, by employing the Ptb and Bukenzymes, we were able to couple the production of ATP and IPA molecules, resulting ina more energetically favourable process for IPA production in E. coli.We cultivated the developed pbu strain in lab-scale bioreactors under N-starved conditions.The higher ATP availability in the pbu strain may have resulted in prioritizing biomass formation over IPA production, leading to a relatively high μmax of 0.22 h−1and onset of IPA production only under growth-arrested N-starved conditions. The IPAtiters reached 2.2 g·L−1 after 43 hours of N-starvation. These titers were 1.5 times higher than the titers reported in the literature. However, they were at least twice lower than the titers achieved by the non-ATP-coupled AtoDA-containing (aDA) strain previously developed by our group. The aDA strain had a slower growth rate but was capable of producing IPA in varying quantities during the entire 120-hour process. Under growth arrested conditions, both pbu and aDA strains exhibited similar specific acetate uptake and IPA production rates.Overall, this work demonstrates the potential of the novel ATP-coupled IPA-producing pathway, IPA ptb buk, implemented in E. coli and encourages its application in future metabolic engineering strategies.