Fig. 2: The scFv antibody titers from screening-positive clones.

Relative titers of REMI-based screening positives cultured in 96-deep-well plates (a) or test tubes (b). A total of 43 positive strains were chosen from the REMI-based screen (a) and for re-evaluation (b) on the wild-type K. phaffii strain. A single highly positive and many weakly positive clones were obtained. The top 20 clones with higher protein secretion performance in test tube cultivation (1.3-times higher than the host strain) were labeled in red (except for 79G10, in which a mutation in the MFα secretion signal peptide contributed to the scFv secretion performance) (b). After identifying the plasmid integration locus for the 20 strains, nine unique gene disruptions were identified. The titer values of screening positives were normalized to that of the host strain. Error bars represent standard deviations from three technical replicates (c). Relative titers of test tube cultivations for dnl4his4–based re-construction strains at 60 h after methanol induction. Among the nine reconstructed strains, six gene-disrupted strains showed higher titers than the host strain with significant difference, indicating that six gene-disruption-type effective factors were identified. d Growth rate analysis of the six gene-disrupted strains and the host strain on BMGY (emerald, diagonal stripes) and BMMY (orange, filled bars) are indicated. e, f The re-constructed gene-disruption and the host strains were cultivated in the shake flasks. The titer (e) and final OD₆₆₀ values (f) of each strain at 24 h (light green, diagonal stripes) and 48 h (dark green, filled bars) sampling points after methanol induction are indicated. Error bars represent standard deviations from three biological replicates. Asterisks and “ns” indicate the significance level (p < 0.05 by t-test) and non-significance level (p > 0.05 by t-test) between the host and effective factor-disruption strains, respectively.