Figure 8

Diploid strain stability in high-productivity fed-batch fermentation. (a) Time course green and red fluorescence intensities of YGLY27431 showing OCH1 wild-type P. pastoris diploid strain stability over 10 days of induction. (b) Time course green and red fluorescence intensities of YGLY27435 showing glycoengineered -P. pastoris diploid strain stability over 10 days of induction. (c) Time-dependent diploid strain stability analyzed using a selection plates assay. (d) PCR confirmation of diploid genome. (e) PCR confirmation of sporulated haploid genome. For a, and b, fluorescence intensities were measured at 24-hour time interval using flow cytometry. For c, percentage of diploid population was determined by the number of colonies growing in diploid selection plate in comparison with the number of colonies growing in non-selective plates. For d, and e, the primer sets listed in Table 3 were used to confirm the existence of anti-HER2 light and heavy chain expression cassettes in yeast chromosome.