Anaerobic batch fermentation profiles of a glucose/xylose mixture.

<p>Fermentation experiments were performed using 2× YNB medium containing 20 g L⁻¹ glucose and 50 g L⁻¹ xylose. Figures show representative values from one experiment out of two biological duplicates using TMB3492 (Hxk2p-wt) (red) and TMB3493 (Hxk2p-Y) (turquoise). <b>A</b>) Fermentation profiles of glucose (▪) and xylose (•) consumption and production of ethanol (♦). <b>B</b>) Fermentation profiles of xylitol (•), glycerol (♦) and biomass (▴) formation.</p

Strains and plasmids used in this study.

a<p>Sequence numbering for some constructs are numbered in relation to the first base in the ATG starting codon of the ORF in question.</p

Selection of Hxk2p variants.

<p>The selection of Hxk2p variants was performed in anaerobic glucose limited chemostat cultivation with feed containing 5 g L⁻¹ glucose and 50 g L⁻¹ xylose. After 96 h of continuous cultivation of TMB3463 transformed with the <i>HXK2</i>-library at <i>D</i> = 0.072 h⁻¹, the dilution rate was increased to <i>D</i> = 0.40 h⁻¹ (indicated as <i>t</i> = 0 h). During the washout the specific growth rate was calculated from the equation d ln(OD) d<i>t</i>⁻¹ =  <i>μ</i>_max−<i>D</i>. The natural logarithm of OD is shown as red squares (▪). The washout profile displays two growth phases and the switching point occurs when the residual glucose concentration (▴) exceeds 3 g L⁻¹. After 10 h the glucose concentration stabilized at 4 g L⁻¹ which reduced the selection pressure by inhibiting the uptake of xylose and thus slowed down the washout of cells. The measured accumulation of glucose was less than the theoretical (dashed line), calculated according to <i>S</i> =  <i>S</i>_in+(<i>S</i>₀−<i>S</i>_in)·e^{(−<i>D</i>·<i>t</i>)}, showing that the consumption was not negligible.</p

Specific glucose phosphorylating activity during xylose-induced inactivation of Hxk2p-wt and Hxk2p-Y.

<p>Specific glucose phosphorylating activity (bars) in strains TMB3466 (Hxk2p-wt) (red colour) and TMB3467 (Hxk2p-Y) (turquoise colour) in anaerobic glucose-limited chemostat cultivations. At steady state (s.s.) on glucose (Glc) the two strains exhibited similar activity but during the accumulation of xylose (Xyl; •) the wild-type enzyme became inhibited faster than the variant. At steady state in the presence of xylose the variant had 64% higher specific activity compared with the wild-type. Specific activities were determined from duplicate biological experiments. At steady state conditions two different samples were collected with at least 2.5 volume changes in between.</p

Mutations included in the condensed Hxk2p library.

<p>The table lists the amino acid residues chosen for mutagenesis, the alternative residues in each position and the codon degeneracy used.</p>a<p>Codon degeneracy: S = C or G; W = A or T; R = A or G; K = G or T.</p