Table 4.

Recombinant gene expression in ancestral and evolved P. pastoris X-33 strains

Strain	rHSA (mg L−1)	Final OD600	rHSA OD−1600	Strain	rFDL (U L−1)	Final OD600	rFDL OD−1600
High biomass deep-well culturesa
X-33 wt	13.8 ± 3.0	7.2 ± 0.2	1.9 ± 0.4	X-33 wt	nd	nd	nd
Y250 2c	0.9 ± 0.3	5.3 ± 0.2	0.2 ± 0.1	Y250 2c	nd	nd	nd
Y250 3a	4.7 ± 0.7	5.7 ± 0.3	0.9 ± 0.1	Y250 3a	nd	nd	nd
M250 1a	17.8 ± 2.0	7.3 ± 0.3	2.5 ± 0.3	M250 1a	nd	nd	nd
M250 3b	15.9 ± 2.9	8.4 ± 0.2	1.9 ± 0.3	M250 3b	nd	nd	nd

Strain	YPM rHSAb			Strain	BMM rHSAb
	rHSA (mg L−1)	Final OD600	rHSA OD−1600		rHSA (mg L−1)	Final OD600	rHSA OD−1600
Low biomass deep-well cultures
X-33 wt	13.8 ± 0.1	4.1 ± 0.2	3.4 ± 0.2	X-33 wt	3.9 ± 0.4	2.8 ± 0.0	1.3 ± 0.1
Y250 2c	0.4 ± 0.1	1.9 ± 0.1	0.2 ± 0.1	Y250 2c	0.7 ± 0.7	0.2 ± 0.0	3.9 ± 3.9
Y250 3a	2.0 ± 0.6	2.8 ± 0.2	0.7 ± 0.2	Y250 3a	0.1 ± 0.0	0.2 ± 0.0	0.6 ± 0.1
M250 1a	16.6 ± 2.0	5.9 ± 1.2	2.8 ± 0.2	M250 1a	7.0 ± 1.5	4.2 ± 0.2	1.6 ± 0.3
M250 3b	24.7 ± 5.7	7.4 ± 0.2	3.4 ± 0.9	M250 3b	4.7 ± 0.9	4.6 ± 0.1	1.0 ± 0.2

Strain	YPM rFDLc			Strain	BMM rFDLc
	rFDL (U L−1)	Final OD600	rFDL OD−1600		rFDL (U L−1)	Final OD600	rFDL OD−1600
Low biomass deep-well cultures
X-33 wt	179.4 ± 27.3	3.9 ± 0.1	38.4 ± 12.5	X-33 wt	8.5 ± 2.0	2.2 ± 0.3	3.5 ± 0.5
Y250 2c	10.3 ± 3.0	1.9 ± 0.0	5.6 ± 1.6	Y250 2c	6.0 ± 1.4	0.2 ± 0.0	36.1 ± 9.8
Y250 3a	16.9 ± 8.4	2.6 ± 0.0	6.7 ± 3.4	Y250 3a	16.4 ± 6.5	0.1 ± 0.0	122.7 ± 49.1
M250 1a	419.5 ± 26.5	3.8 ± 0,1	108.7 ± 7.5	M250 1a	65.0 ± 5.5	2.5 ± 0.1	25.6 ± 1.5
M250 3b	448.0 ± 57.3	4.1 ± 0.0	109.5 ± 13.9	M250 3b	40.0 ± 5.6	2.5 ± 0.1	17.8 ± 2.2

rHSA and rFDL were used as model proteins for expression in deep well cultures. For rHSA two screening protocols with high and low starting biomass prior to induction and growth on methanol were applied. For rFDL the low starting biomass protocol was applied. ^a and ^b number of replicates n = 12; ^c n = 6. Values represent averages ± standard deviation