Engineering yeast hexokinase 2 for improved tolerance toward xylose-induced inactivation

Abstract

Hexokinase 2 (Hxk2p) from Saccharomyces cerevisiae is a bi-functional enzyme being both a catalyst and an important regulator in the glucose repression signal. In the presence of xylose Hxk2p is irreversibly inactivated through an autophosphorylation mechanism, affecting all functions. Consequently, the regulation of genes involved in sugar transport and fermentative metabolism is impaired. The aim of the study was to obtain new Hxk2p-variants, immune to the autophosphorylation, which potentially can restore the repressive capability closer to its nominal level. In this study we constructed the first condensed, rationally designed combinatorial library targeting the active-site in Hxk2p. We combined protein engineering and genetic engineering for efficient screening and identified a variant with Phe159 changed to tyrosine. This variant had 64% higher catalytic activity in the presence of xylose compared to the wild-type and is expected to be a key component for increasing the productivity of recombinant xylose-fermenting strains for bioethanol production from lignocellulosic feedstocks.

Figure 1. Selection of Hxk2p variants.

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 HXK2-library at D = 0.072 h⁻¹, the dilution rate was increased to D = 0.40 h⁻¹ (indicated as t = 0 h). During the washout the specific growth rate was calculated from the equation d ln(OD) dt ⁻¹ =  μ _max−D. 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 S =  S _in+(S ₀−S _in)·e^(−D·t), showing that the consumption was not negligible.

Figure 2. Specific glucose phosphorylating activity during xylose-induced inactivation of Hxk2p-wt and Hxk2p-Y.

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.

Figure 3. Anaerobic batch fermentation profiles of a glucose/xylose mixture.

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). A) Fermentation profiles of glucose (▪) and xylose (•) consumption and production of ethanol (♦). B) Fermentation profiles of xylitol (•), glycerol (♦) and biomass (▴) formation.

Figure 4. Homology models of the Hxk2p active site.

The key residues in the active site of Hxk2p that selected for mutagenesis are displayed with corresponding label (see also Table 2). The position and orientation of the Phe159 and Tyr159 residues are shown in A) and B), respectively. Both figures show the orientation of the β-d-glucopyranose structure (yellow colour) in the active site.