Evolution-assisted engineering of E. coli improved succinic acid production from glycerol

Glycerol, a major byproduct of biodiesel production, serves as a promising and versatile feedstock for synthesizing high-value chemicals. In this study, the NZN111, a potential succinic acid (SA) producer, was subjected to adaptive laboratory evolution (ALE) under selective pressure imposed by sodium acetate (NaAC) pressure. By analyzing the differences in the transcript levels of genes related to substrate utilization and product generation in the evolved strains, it was found that NaAC metabolism in <i>Escherichia coli</i> involves a series of enzymatic reactions that are apparently benefit for glycerol metabolism and SA biosynthesis. To further enhance the strain's performance, a metabolic engineering strategy was implemented, including introduction of exogenous carboxykinases and HCO₃ ^- transporter proteins. The engineered strain produced 84.27 ​g/L SA in an anaerobic fermentation medium containing 100 ​g/L glycerol with a yield of 1.25 ​g/g glycerol, which was 1.38 and 1.16 times higher than those of the original strain, respectively. These findings highlight the potential of combining ALE with targeted metabolic engineering to develop robust microbial platforms for the efficient conversion of glycerol into high-value chemicals, contributing to the sustainable utilization of biodiesel byproducts.