Isobutyraldehyde production from Escherichia coli by removing aldehyde reductase activity

We adopted the isobutanol pathway previously constructed in E. coli, neglecting the last step in the pathway where isobutyraldehyde is converted to isobutanol. However, this strain still overwhelmingly produced isobutanol (1.5？g/L/OD600 (isobutanol) vs 0.14？g/L/OD600 (isobutyraldehyde)). Next, we deleted yqhD which encodes a broad-substrate range aldehyde reductase known to be active toward isobutyraldehyde. This strain produced isobutanol and isobutyraldehyde at a near 1:1 ratio, indicating further native isobutyraldehyde reductase (IBR) activity in E. coli. To further eliminate isobutanol formation, we set out to identify and remove the remaining IBRs from the E. coli genome. We identified 7 annotated genes coding for IBRs that could be active toward isobutyraldehyde: adhP, eutG, yiaY, yjgB, betA, fucO, eutE. Individual deletions of the genes yielded only marginal improvements. Therefore, we sequentially deleted all seven of the genes and assessed production. The combined deletions greatly increased isobutyraldehyde production (1.5？g/L/OD600) and decreased isobutanol production (0.4？g/L/OD600). By assessing production by overexpression of each candidate IBR, we reveal that AdhP, EutG, YjgB, and FucO are active toward isobutyraldehyde. Finally, we assessed long-term isobutyraldehyde production of our best strain containing a total of 15 gene deletions using a gas stripping system with in situ product removal, resulting in a final titer of 35？g/L after 5？days.In this work, we optimized E. coli for the production of the important chemical feedstock isobutyraldehyde by the removal of IBRs. Long-term production yielded industrially relevant titers of isobutyraldehyde with in situ product removal. The mutational load imparted on E. coli in this work demonstrates the versatility of metabolic engineering for strain improvements.