Adaptive evolution of Escherichia coli inactivated in the phosphotransferase system operon improves co-utilization of xylose and glucose under anaerobic conditions

Abstract

Modification of the phosphoenolpyruvate/sugar phosphotransferase system (PTS) has shown improvement in sugar coassimilation in Escherichia coli production strains. However, in preliminary experiments under anaerobic conditions, E. coli strains with an inactive PTS and carrying pLOI1594, which encodes pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis, were unable to grow. These PTS⁻ strains were previously evolved under aerobic conditions to grow rapidly in glucose (PTS⁻ Glucose+ phenotype). Thus, in this work, applying a continuous culture strategy under anaerobic conditions, we generate a new set of evolved PTS⁻ Glucose+ mutants, VH30N1 to VH30N6. Contrary to aerobically evolved mutants, strains VH30N2 and VH30N4 carrying pLOI1594 grew in anaerobiosis; also, their growth capacity was restored in a 100%, showing specific growth rates (μ ~ 0.12 h⁻¹) similar to the PTS+ parental strain (μ = 0.11 h⁻¹). In cultures of VH30N2/pLOI1594 and VH30N4/pLOI1594 using a glucose-xylose mixture, xylose was totally consumed and consumption of sugars occurred in a simultaneous manner indicating that catabolic repression is alleviated in these strains. Also, the efficient sugar coassimilation by the evolved strains caused an increment in the ethanol yields.