Engineering and laboratory evolution of Pseudomonas putida KT2440 to utilize lactose as a carbon source

During the last years, metabolic engineering has greatly expanded the scope of different valuable chemicals that can be synthesized or degraded with the help of genetically modified microorganisms. The soil bacterium Pseudomonas putida, especially the KT2440 strain, is emerging as a new favourite host for biotransformation of both industrial and environmental interest due to its capability to withstand various physicochemical stresses, its rapid growth and versatile metabolism, non-pathogenicity, and the availability of molecular tools for advanced genetic programming. 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 beta-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 that were 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.