High-level heterologous production of propionate in engineered Escherichia coli.

Previously, we derived a propanologenic (i.e., 1-propanol-producing) bacterium Escherichia coli strain by activating the genomic Sleeping beauty mutase (Sbm) operon. The activated Sbm pathway branches out of the tricarboxylic acid (TCA) cycle at the succinyl-CoA node to form propionyl-CoA and its derived metabolites of 1-propanol and propionate. In this study, we targeted several TCA cycle genes encoding enzymes near the succinyl-CoA node for genetic manipulation in order to identify the individual contribution of the carbon flux into the Sbm pathway from the three TCA metabolic routes, i.e., oxidative TCA cycle, reductive TCA branch, and glyoxylate shunt. For the control strain CPC-Sbm in which propionate biosynthesis occurred under relatively anaerobic conditions, the carbon flux into the Sbm pathway were primarily derived from the reductive TCA branch; and both succinate availability and the SucCD-mediated interconversion of succinate/succinyl-CoA were critical for such carbon flux redirection. While the oxidative TCA cycle normally had a minimal contribution to the carbon flux redirection, the glyoxylate shunt could be an alternative and effective carbon flux contributor under aerobic conditions. With mechanistic understanding of such carbon flux redirection, metabolic strategies based on blocking the oxidative TCA cycle (via ∆sdhA mutation) and deregulating the glyoxylate shunt (via ∆iclR mutation) were developed to enhance the carbon flux redirection and, therefore, propionate biosynthesis, achieving a high propionate titer of 30.9 g l-1 with an overall propionate yield of 49.7% upon fed-batch cultivation of the double mutant strain CPC-Sbm∆sdhA∆iclR under aerobic conditions. The results also suggest that the Sbm pathway could be metabolically active under both aerobic and anaerobic conditions. This article is protected by copyright. All rights reserved.