Knockout of acetoacetate degradation pathway gene atoDA enhances the toxicity tolerance of Escherichia coli to isopropanol and acetone.

Abstract

Isopropanol and acetone are important chemical products and potential high-quality new fuels. Both of them are metabolites of isopropanol synthesis pathway, but they are toxic to most bacteria. In this study, toxicity tolerance of Escherichia coli strains was evaluated by detecting their growth rates under different concentrations of isopropanol and acetone. It was showed that isopropanol was more toxic to E. coli than acetone, and the native strain MG1655 had better tolerance over DH5α to either acetone or isopropanol of 300mM. Key genes of ethanol synthesis pathway, acetic acid metabolism pathway, and acetoacetic acid degradation pathway, including adhE, ackA-pta, and atoDA, were knocked out in MG1655 to form mutants MGΔadhE, MGΔackA-pta, and MGΔatoDA. The tolerance performances of the mutants to isopropanol and acetone were determined under various concentrations including 300mM, 500mM, and 700mM, respectively. The mutant MGΔatoDA exhibited excellent tolerance to both acetone and isopropanol of 500mM, and MGΔackA-pta could tolerate acetone at 500mM rather than isopropanol, while the deletion of adhE in MGΔadhE resulted in a severe cell growth defection. Although isopropanol and acetone at 700mM caused severe growth inhibition on each strain, cell growth could be restored to varying degrees with the prolongation of culture time. This phenomenon was suggested to be related to the volatilization of isopropanol and acetone based on volatilization tests. It was envisioned that MG1655 was a suitable host strain for isopropanol metabolic engineering research, and the acetoacetic acid degradation pathway gene atoDA, was probably the key optimizing point for isopropanol production.