Engineering and laboratory evolution of lactose-utilizing Pseudomonas putida strains

Pseudomonas putida strain KT2440 has been extensively adopted for bioengineering purposes. Despite of its versatile metabolism, this strain is not able to utilize lactose as a carbon source. In this study, we report the construction of KT2440 derivative that can utilize this disaccharide as a growth substrate. The metabolic engineering of this strain was accompanied with the laboratory evolution of bacteria on lactose minimal media. Specifically, the expression of β-galactosidase gene lacZ on a plasmid was accompanied with integration of galactose Leloir pathway genes galETKM and lactose permease gene lacY into the chromosome of KT2440. Thereafter, the engineered strain KT2440Gal was adapted on minimal medium containing lactose as an only carbon source. Three lactose-utilizing mutants were further characterized for their growth on lactose and other sugars, and mutations required to utilize lactose were confirmed by reverse engineering. This study has expanded a potential use of P. putida KT2440 in bioprocesses that are relayed on utilization of various sugars.