Alleviating our society’s dependence on petroleum-based chemicals has been highly emphasized due to fossil fuel shortages and increasing greenhouse gas emissions. Isopropanol is a molecule of high potential to replace some petroleum-based chemicals, which can be produced through biological platforms from renewable waste carbon streams such as carbohydrates, fatty acids, or CO₂. In this study, for the first time, the heterologous expression of engineered isopropanol pathways were evaluated in a Cupriavidus necator strain Re2133, which was incapable of producing poly-3-hydroxybutyrate [P(3HB)]. These synthetic production pathways were rationally designed through codon optimization, gene placement, and gene dosage in order to efficiently divert carbon flow from P(3HB) precursors toward isopropanol. Among the constructed pathways, Re2133/pEG7c overexpressing native C. necator genes encoding a β-ketothiolase, a CoA-transferase, and codon-optimized Clostridium genes encoding an acetoacetate decarboxylase and an alcohol dehydrogenase produced up to 3.44 g l^-1 isopropanol in batch culture, from fructose as a sole carbon source, with only 0.82 g l^-1 of biomass. The intrinsic performance of this strain (maximum specific production rate 0.093 g g^-1 h^-1, yield 0.32 Cmole Cmole^-1) corresponded to more than 60 % of the respective theoretical performance. Moreover, the overall isopropanol production yield (0.24 Cmole Cmole^-1) and the overall specific productivity (0.044 g g^-1 h^-1) were higher than the values reported in the literature to date for heterologously engineered isopropanol production strains in batch culture. Strain Re2133/pEG7c presents good potential for scale-up production of isopropanol from various substrates in high cell density cultures.

Summary: Formic acid, acting as both carbon and energy source, is a safe alternative to a carbon dioxide, hydrogen and dioxygen mix for studying the conversion of carbon through the C alvin– B enson– B assham ( CBB ) cycle into value‐added chemical compounds by non‐photosynthetic microorganisms. In this work, organoautotrophic growth of R alstonia eutropha on formic acid was studied using an approach combining stoichiometric modeling and controlled cultures in bioreactors. A strain deleted of its polyhydroxyalkanoate production pathway was used in order to carry out a physiological characterization. The maximal growth yield was determined at 0.16 C mole C mole ⁻¹ in a formate‐limited continuous culture. The measured yield corresponded to 76% to 85% of the theoretical yield (later confirmed in pH ‐controlled fed‐batch cultures). The stoichiometric study highlighted the imbalance between carbon and energy provided by formic acid and explained the low growth yields measured. Fed‐batch cultures were also used to determine the maximum specific growth rate (μ _max = 0.18 h ⁻¹ ) and to study the impact of increasing formic acid concentrations on growth yields. High formic acid sensitivity was found in R eutropha since a linear decrease in the biomass yield with increasing residual formic acid concentrations was observed between 0 and 1.5 g l ⁻¹ .