Enhanced N-Butanol Tolerance of E. Coli Via Adaptive Evolution

Synthetic biology provides a significant platform in creating novel pathways/organisms for producing useful compounds, while it remains a challenge to enhance the production efficiency. Recently we constructed a recombinant Escherichia coli for glutarate production using a synthetic α-ketoacid reduction pathway, in which α-ketoglutarate is reduced to 2-hydroxyglutarate then converted to glutarate. However, the production titer was low, which may be due to 1) oxygen-sensitive nature of 2-hydroxyglutaryl-CoA dehydratase (HgdABC) and 2) limited cell growth in anaerobic cultivation. Therefore, we developed an aerobic-anaerobic two-stage strategy by growing more cells aerobically, then shifting to anaerobic cultivation to ensure the functional HgdABC for glutarate biosynthesis. The two-stage cultivation resulted in higher production of glutarate and other two C5 dicarboxylic acids – glutaconate and 2-hydroxylglutarate than the original anaerobic process. Furthermore, we used an anaerobically-inducible nar promoter to improve the hgdABC expression responding to aerobic-anaerobic shift. Finally, the glutarate, glutaconate and 2-hydroxyglutarate titer was increased about 2, 5 and 3 times, reaching 11.6, 108.8 and 399.5 mg/L, respectively. The work demonstrated an effective strategy for ameliorating α-ketoacid reduction pathway to produce C5 dicarboxylic acids, as well as the potential of integration of bioprocess and metabolic engineering for enhancing chemicals production by an engineered microorganism.