Fig. 3: Combined perturbation of gene-disruption-type effective factors in a single strain.

a, b The scFv titer (a) and the productivity (b) (scFv titer per OD₆₆₀ value) in shake flask cultivation at 24 h (diagonal stripes), 48 h (filled bars) and 72 h sampling (vertical stripes) after methanol induction are indicated. For each timepoint, scFv productivity was normalized to that of the host strain at 24 h. The scFv productivity of the multi-deletion strains (RD1 + 2, RD1 + 2 + 3 and RD1 + 2 + 3 + 4) were significantly higher than that of the single effective factor-disruption and the host strains. 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 RD-2 strain (the best secretion strain among single effective factor disruptions) and the accumulated strains at 48 h methanol induction, respectively. c, d Effects of swapping of the secreted protein and promoter in combined gene-disruption-type effective factor strains. Relative productivities (BGL1p activity or scFv titer per final OD₆₆₀ value) of host and gene-disruption(s) strains in test tube cultivation for 60 h are indicated. (c) AOX1 promoter-driven BGL1 and (d) GAPDH constitutive promoter-driven anti-lysozyme scFv constructs are indicated. Each productivity value was normalized to that of the host strain. Gray, blue, red, purple, yellow, yellow-green, orange and blue-green indicate the host, RD-1, RD-2, RD-3, RD-4, RD1 + 2, RD1 + 2 + 3 and RD1 + 2 + 3 + 4 strains, respectively. Error bars represent standard deviation from three biological replicates. Asterisks indicate that a significant difference was observed (p < 0.05 by t-test) in the BD-2 or GD-1 strain (the best secretion strains among single effective factor disruptions) and the multi-gene-disrupted strains.