Enhancing the production of isopropanol with reduced CO 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 CO 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 CO emission, carbonic anhydrase from Hydrogenovibrio marinus (HmCA) and acetoacetyl-CoA synthase (nphT7) were overexpressed, constructing CGIPA-5 strain, enabling conversion of CO 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 IPA, with CO emission reduced by 30 % compared to CGIPA-4 strain. This work demonstrates a sustainable approach to petrochemical replacement through protein and metabolic engineering.