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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.

SUBMITTER: Bergdahl B

PROVIDER: S-EPMC3765440 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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

Bergdahl Basti B Sandström Anders G AG Borgström Celina C Boonyawan Tarinee T van Niel Ed W J EW Gorwa-Grauslund Marie F MF

PloS one 20130906 9

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 potentiall ...[more]

PMID: 24040384

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Project description:BackgroundStress tolerance is one of the important desired microbial traits for industrial bioprocesses, and global regulatory protein engineering is an efficient approach to improve strain tolerance. In our study, IrrE, a global regulatory protein from the prokaryotic organism Deinococcus radiodurans, was engineered to confer yeast improved tolerance to the inhibitors in lignocellulose hydrolysates or high temperatures.ResultsThree IrrE mutations were developed through directed evolution, and the expression of these mutants could improve the yeast fermentation rate by threefold or more in the presence of multiple inhibitors. Subsequently, the tolerance to multiple inhibitors of single-site mutants based on the mutations from the variants were then evaluated, and 11 mutants, including L65P, I103T, E119V, L160F, P162S, M169V, V204A, R244G, Base 824 Deletion, V299A, and A300V were identified to be critical for the improved representative inhibitors, i.e., furfural, acetic acid and phenol (FAP) tolerance. Further studies indicated that IrrE caused genome-wide transcriptional perturbation in yeast, and the mutant I24 led to the rapid growth of Saccharomyces cerevisiae by primarily regulating the transcription level of transcription activators/factors, protecting the intracellular environment and enhancing the antioxidant capacity under inhibitor environments, which reflected IrrE plasticity. Meanwhile, we observed that the expression of the wild-type or mutant IrrE could also protect Saccharomyces cerevisiae from the damage caused by thermal stress. The recombinant yeast strains were able to grow with glucose at 42 ℃.ConclusionsIrrE from Deinococcus radiodurans can be engineered as a tolerance-enhancer for Saccharomyces cerevisiae. Systematic research on the regulatory model and mechanism of a prokaryotic global regulatory factor IrrE to increase yeast tolerance provided valuable insights for the improvements in microbial tolerance to complex industrial stress conditions.

Dataset Information

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

Publications

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

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