(175ab) Optimizing Cellobiose Consumption of Escherichia coli by Metabolic Engineering and Adaptive Laboratory Evolution

The industrial strain Escherichia coli is known to prefer D-glucose as substrate over other biomass-derived sugars like D-xylose, L-arabinose, and D-galactose. This natural behavior of E. coli is known as the carbon catabolite repression (CCR). Circumventing this bacterial phenotype has been deemed important by bioengineers since simultaneous consumption of multiple sugar substrates greatly improve the productivity in microbial bioprocesses. One way to overcome CCR is to replace D-glucose with cellobiose, a D-glucose disaccharide. Since E. coli is not equipped with the enzymes to degrade cellobiose, it is necessary to recruit and overexpress cellobiose hydrolases, which break down cellobiose into its monomeric D-glucose units inside the cell. In this work, an alternative enzyme called cellobiose phosphorylase from Saccharophagus degradans was cloned and overexpressed in E. coli. This enzyme is encoded by cep94A gene and it releases D-glucose and D-glucose-1-phosphate as the enzyme reaction products inside the cell. The resulting recombinant strain, ECcepA, can grow on cellobiose as a sole carbon source with a growth rate of 0.32 h^-1 while the same strain has a growth rate of 0.51 h^-1 when grown on D-glucose. The growth rate of ECcepA was improved by employing adaptive laboratory evolution strategy in minimal media with 4 g L^-1 cellobiose as carbon source. After 600 generations (or 41 days), the evolved strain, ECcepA*, has a growth rate of 0.66 h^-1. The full genome of ECcepA* was sequenced to pinpoint the location of mutations that conferred the improved growth phenotype on cellobiose. These mutations were then replicated in the parental strain ECcepA to confirm the role of these mutations on the improved growth of ECcepA*. Furthermore, CCR was not observed in the evolved strain when it was grown on cellobiose with D-xylose, L-arabinose or D-galactose. Overall, utilizing cellobiose as an alternative to D-glucose as substrate effectively bypasses CCR in E. coli. This strategy includes the overexpression of a cellobiose phosphorylase and the identified set of mutations in the genome of the host, which altogether led to an optimized cellobiose-consuming strain.

This work was supported by Korea Research Fellowship Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2015H1D3A1062172) and by the Ministry of Education (2018R1D1A1B07043993 and 22A20130012051(BK21Plus)).