Genome shuffling of Lactobacillus brevis for enhanced production of thymidine phosphorylase

Thymidine phosphorylase (TPase) plays a vital role in the biosynthesis of nucleosides and their analogs which have tremendous potential in antiviral and anticancer therapies. In this study, genome shuffling was applied to develop new strains of Lactobacillus brevis with an enhanced production of TPase. The parent organisms were mutated using ultraviolet (UV) irradiation and were shuffled by recursive pool-wise protoplast fusion. The parent protoplasts of each cycle were inactivated by UV irritation for 50 min or by heating at 60°C for 60 min. A rapid and efficient pre-screening method for determining L. brevis fusants with increased TPase production was established by adding appropriate concentrations of substrate thymidine and potassium phosphate to the culture broth based on significant differences in the absorption spectra of substrate thymidine and its product, thymine, in alkaline solution at 290 nm. Strains F3-19 and F3-36 showed high TPase activity and favorable hereditary stability and were screened out through three rounds of recursive protoplast fusion. The increase in the TPase activity of F3-19 and F3-36 was 252.6 and 260.5%, respectively, in comparison with the wild type.