Isobutyraldehyde production from <it>Escherichia coli</it> by removing aldehyde reductase activity

• Main Authors: Rodriguez Gabriel M, Atsumi Shota
• Format: Article
• Language: English
• Published: BMC 2012-06-01
• Series: Microbial Cell Factories
• Online Access: http://www.microbialcellfactories.com/content/11/1/90

<p>Abstract</p> <p>Background</p> <p>Increasing global demand and reliance on petroleum-derived chemicals will necessitate alternative sources for chemical feedstocks. Currently, 99% of chemical feedstocks are derived from petroleum and natural gas. Renewable methods for producing important chemical feedstocks largely remain unaddressed. Synthetic biology enables the renewable production of various chemicals from microorganisms by constructing unique metabolic pathways. Here, we engineer <it>Escherichia coli</it> for the production of isobutyraldehyde, which can be readily converted to various hydrocarbons currently derived from petroleum such as isobutyric acid, acetal, oxime and imine using existing chemical catalysis. Isobutyraldehyde can be readily stripped from cultures during production, which reduces toxic effects of isobutyraldehyde.</p> <p>Results</p> <p>We adopted the isobutanol pathway previously constructed in <it>E. coli</it>, 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 <it>yqhD</it> 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 <it>E. coli</it>. To further eliminate isobutanol formation, we set out to identify and remove the remaining <it>IBR</it>s from the <it>E. coli</it> genome. We identified 7 annotated genes coding for IBRs that could be active toward isobutyraldehyde: <it>adhP</it>, <it>eutG</it>, <it>yiaY</it>, <it>yjgB</it>, <it>betA</it>, <it>fucO</it>, <it>eutE</it>. 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 <it>IBR</it>, 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 <it>in situ</it> product removal, resulting in a final titer of 35 g/L after 5 days.</p> <p>Conclusions</p> <p>In this work, we optimized <it>E. coli</it> for the production of the important chemical feedstock isobutyraldehyde by the removal of IBRs. Long-term production yielded industrially relevant titers of isobutyraldehyde with <it>in situ</it> product removal. The mutational load imparted on <it>E. coli</it> in this work demonstrates the versatility of metabolic engineering for strain improvements.</p>