Abstract: By constructing the targeting vector of the uracil phosphoribosyl transferase gene (upp), the upp gene deletion mutant K. vulgare Δupp was obtained using homologous recombination. The mutant was selected as the chassis cell in seamlesss genome modification system. An upp gene expression vector was constructed and transformed into mutant K. vulgare Δupp. After the transformation, an upp gene reverse mutation strain named PBB-upp was obtained, and the feasibility of upp as a negative selectable marker was verified. The results showed that Δupp mutants rather than the wildtype and reverse PBB-upp strains could grow in a medium with 1 mg/mL 5-fluorouracil. Moreover, there were significant differences among the three strains in terms of 5-fluorouracil resistance, indicating that by combining the upp gene as a negative marker with a positive marker, double homologous recombinations could occur on the chassis cell without the upp gene. In this way, the modification can be achieved without antibiotic marker in Ketogulonigenium vulgare.

Key words: Ketogulonigenium vulgare, negative selection marker, scarless gene alteration, 5-fluorouracil