Enhancing the production of isopropanol with reduced CO₂ emission via protein and metabolic engineering using Corynebacterium glutamicum

Isopropanol (IPA), a versatile chemical with applications in various fields of industries, yet its petroleum-based production raises environmental concerns. In this study, Corynebacterium glutamicum was engineered to enhance IPA production while mitigating CO₂ emissions. First, the rational design of secondary alcohol dehydrogenase (SADH) variants with shifted cofactor specificity from NADPH to NADH, resulting in an 11.11-fold increased NADH oxidation rate and 6.02-fold increased enzyme activity. Secondly, modified SADH was used in combination with the Ncgl1676 promoter in order to separate the growth and production phases. This engineering resulted in a strain called CGIPA-4, which showed a 2.45-fold increase in IPA production. To address CO₂ emission, carbonic anhydrase from Hydrogenovibrio marinus (HmCA) and acetoacetyl-CoA synthase (nphT7) were overexpressed, constructing CGIPA-5 strain, enabling conversion of CO₂ into bicarbonate, which supported IPA biosynthesis and reduced emissions by up to 21 %. Finally, high cell density fed-batch fermentation using CGIPA-5 strain produced 148.6 ± 3.8 g L⁻¹ IPA, with CO₂ emission reduced by 30 % compared to CGIPA-4 strain. This work demonstrates a sustainable approach to petrochemical replacement through protein and metabolic engineering.