Fig. 2

Markerless genome editing in P. pastoris using mazF-zeoR counter-selection approach. A Schematic illustrating the work flow of markerless genome editing system using mazF-zeoR counter-selection approach. Three homology arms with different lengths (up-arm and down-arm, ~ 1000–1200 bp; short-arm, ~ 100–300 bp) were used for gene deletion or integration. Two screening steps including two rounds of homologous recombination are involved. In step 1, the digested plasmid was transformed into P. pastoris under the selection of antibiotic zeocin, allowing the integration of “mazF-zeocin-short-arm” fragment onto the chromosome and the elimination of the target gene. In step 2, the obtained transformants were cultivated in BMMY medium containing methanol as the sole carbon source to induce the expression of mazF gene. The MazF selection stress promotes the removal of “mazF-zeocin” cassette through the second round of homologous recombination. B PCR analysis of JQ01 mutant after each selection step. C, D PCR analysis of JQ02 mutant after each selection step. M, 1-kb DNA ladder; WT, genomic DNA of P. pastoris GS115 as the control; Mut1, mutant strain after first round of recombination; Mut2, mutant strain after second round of recombination