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Table 1 Characteristics of high-yielding yeast strains

From: Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin

Yeast strain

Fps1 yield (µg/L)

FPS1-HA3

5′∆1-43-

FPS1-HA3

5′∆1-215-

FPS1-HA3

M:P ratio

GCN4 expression

Constitutive phosphorylation of eIF2α

Wild-type (BY4741)

0.02 (0.01)

0.21 (0.06)

12.14 (1.68)

0.15 (0.01)

1.0 (0.4)

N

Wild-type + 0.5 µg/mL doxycycline

0.02 (0.01)

–

–

0.15 (0.01)

–

–

yTHCBMS1

0.12 (0.02)

–

–

0.24 (0.03)

2.7 (0.1)

–

yTHCBMS1 + 0.5 µg/mL doxycycline

0.73 (0.15)

2.39 (0.54)

8.00 (2.50)

0.43 (0.14)

2.2 (0.3)

Y

srb5Δ

0.13 (0.03)

–

–

0.39 (0.07)

1.5 (0.1)

Y

gcn5Δ

0.23 (0.04)

–

–

0.16 (0.02)

6.0 (0.5)

Y

spt3Δ

0.91 (0.07)

1.36 (0.28)

4.70 (1.11)

0.18 (0.03)

4.1 (0.4)

Y

  1. Yeast cells were transformed with a plasmid expressing FPS1-HA3, 5′∆1-43-FPS1-HA3 or 5′∆1-215-FPS1-HA3, as indicated. Single transformants were cultured in shake flasks in 2× CBS lacking histidine to maintain the plasmid. Cells were harvested just before the diauxic shift by monitoring residual glucose concentration (cultures had a typical biomass yield of 0.9 g/L; A600 ~ 4). Fps1 yields (µg/L) were determined with reference to a BSA standard curve [38]; a yield of 12.14 µg/L may also be expressed as 13.49 µg/g dry cell weight. The ratio of monosome to polysome peaks (M:P) was calculated from polysome profiles obtained by fractionation on a 10–50% sucrose gradient. To determine GCN4 expression, yeast cells were transformed with plasmid B1805 [31] or, for yTHCBMS1, B1805-HIS. Yeast cells expressing GCN4-LacZ were cultured in shake flasks, harvested at A600 ~ 1 and β-galactosidase levels were determined using ONPG. β-Galactosidase levels are expressed relative to wild-type control cells. For analysis of eIF2α phosphorylation, cells were cultured in the absence (amino-acid-starved cells) or presence (control cells) of amino acids and cells lysates were analysed by immunoblot using anti-eIF2α and anti-phospho-eIF2α antibodies as previously described [39]. All data shown are the mean of biologically-independent, triplicate determinations; where relevant, the standard error of the mean (SEM) is shown in parentheses
  2. Y indicates yes, N indicates no and – indicates that the experiment was not performed
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Microbial Cell Factories

ISSN: 1475-2859

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