Here, we demonstrate that the two major pathways for degradation of serine can be deleted in E. coli MG1655. In addition to above reaction, the conversion of serine to pyruvate (encoded by sdaA, sdaB and tdcG) was also deleted. The resulting strain turned out to be susceptible to even low concentrations of serine in the media. In order to improve the tolerance of the strain towards serine, adaptive laboratory evolution was implemented using a state of the art robotics platform. The strain was grown under inhibiting concentrations of serine in minimal media and was periodically transferred to new media during mid-log phase. After achieving a desired increase in growth rate, the concentration was serine was gradually increased. During the evolution experiment, the serine tolerance was increased substantially. Genome re-sequencing was subsequently used to analyze the genotype of a number of selected strains. These results reveal insights towards the adaptation process as well as the mechanism of serine tolerance.