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.

Real-time monitoring of the sugar sensing in Saccharomyces cerevisiae indicates endogenous mechanisms for xylose signaling

“…Xylose has in previous studies commonly been supplied to engineered S. cerevisiae strains in high concentration (50 g/L) in order to improve the uptake rate, as only unspecific pentose transporters exists in this species [12, 68]. Consequently, 50 g/L xylose was used to screen the strains for any signaling response to this pentose sugar.…”

Xylose-induced dynamic effects on metabolism and gene expression in engineered Saccharomyces cerevisiae in anaerobic glucose-xylose cultures

“…Thus, xylose directly impairs the expression of high-affinity sugar transporter-encoding genes, and the induction of carbon catabolite repression is considered necessary for high-yield fermentation. Recently, a mutant variant of Hxk2p with an increased catalytic activity in the presence of xylose was identified (Bergdahl et al 2013). However, since a S. cerevisiae strain expressing the variant hexokinase 2 had a lower specific xylose uptake rate, Bergdahl and co-workers (Bergdahl et al 2013) speculated that hexokinase 2 may also repress some endogenous activities of S. cerevisiae involved in xylose catabolism (Toivari et al 2004).…”

In-silico smart library design to engineer a xylose-tolerant hexokinase variant

“…Such a high percentage does not recommend mutation of such position and therefore was excluded from the proposed library (Table 3). Bergdahl et al (2013) engineered ScHxk2 targeting selected residues in the glucose binding site. Comparing our library (Tables 3) to the results obtained by Bergdahl et al, showed interesting observations.…”