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Comparative proteomic analysis of a new adaptive Pichia Stipitis strain to furfural, a lignocellulosic inhibitory compound.

ABSTRACT: The development of inhibitor-tolerant ethanologenic yeast is one of the most significant challenges facing bio-ethanol production. Adaptation of Pichia stipitis to inhibitors is one of the most efficient ways for dealing with inhibitor problems. The molecular mechanisms involved in the tolerance and adaptation of P. stipitis are, however, still unclear. In the present study, we developed a yeast strain from P. stipitis Y7 that has improved tolerance against inhibitors. We performed comparative proteomic investigations with sodium dodecyl sulfate polyacrylamide gel electrophoresis and quadrupole time-of-flight mass spectrometry. These investigations gave insights into the tolerance of yeast strains to biomass conversion inhibitors at the protein level. Many proteins involved in glycolysis, the pentose phosphate pathway, and the tricarboxylic acid (TCA) cycle were found to be differentially expressed due to the presence of furfural. Quantitative real-time reverse transcription-PCR (RT-PCR) and metabolite analysis were utilized to provide orthogonal evidence for the results obtained. Our results provide a deeper understanding of the molecular mechanisms involved in the response of P. stipitis to furfural. These findings will benefit the design and development of inhibitor-tolerant yeast.

SUBMITTER: Hao XC

PROVIDER: S-EPMC3621497 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Comparative proteomic analysis of a new adaptive Pichia Stipitis strain to furfural, a lignocellulosic inhibitory compound.

Hao Xue-Cai XC Yang Xiu-Shan XS Wan Ping P Tian Shen S

Biotechnology for biofuels 20130304 1

The development of inhibitor-tolerant ethanologenic yeast is one of the most significant challenges facing bio-ethanol production. Adaptation of Pichia stipitis to inhibitors is one of the most efficient ways for dealing with inhibitor problems. The molecular mechanisms involved in the tolerance and adaptation of P. stipitis are, however, still unclear. In the present study, we developed a yeast strain from P. stipitis Y7 that has improved tolerance against inhibitors. We performed comparative p ...[more]

PMID: 23452435

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Project description:BACKGROUND: The NAD(P)H-dependent Pichia stipitis xylose reductase (PsXR) is one of the key enzymes for xylose fermentation, and has been cloned into the commonly used ethanol-producing yeast Saccharomyces cerevisiae. In order to eliminate the redox imbalance resulting from the preference of this enzyme toward NADPH, efforts have been made to alter the coenzyme specificity of PsXR by site-directed mutagenesis, with limited success. Given the industrial importance of PsXR, it is of interest to investigate further ways to create mutants of PsXR that prefers NADH rather than NADPH, by the alternative directed evolution approach. RESULTS: Based on a homology model of PsXR, six residues were predicted to interact with the adenine ribose of NAD(P)H in PsXR and altered using a semi-rational mutagenesis approach (CASTing). Three rounds of saturation mutagenesis were carried to randomize these residues, and a microplate-based assay was applied in the screening. A best mutant 2-2C12, which carried four mutations K270S, N272P, S271G and R276F, was obtained. The mutant showed a preference toward NADH over NADPH by a factor of about 13-fold, or an improvement of about 42-fold, as measured by the ratio of the specificity constant kcat/Kmcoenzyme. Compared with the wild-type, the kcatNADH for the best mutant was only slightly lower, while the kcatNADPH decreased by a factor of about 10. Furthermore, the specific activity of 2-2C12 in the presence of NADH was 20.6 U.mg-1, which is highest among PsXR mutants reported. CONCLUSION: A seemingly simplistic and yet very effective mutagenesis approach, CASTing, was applied successfully to alter the NAD(P)H preference for Pichia stipitis xylose reductase, an important enzyme for xylose-fermenting yeast. The observed change in the NAD(P)H preference for this enzyme seems to have resulted from the altered active site that is more unfavorable for NADPH than NADH in terms of both Km and kcat. There are potentials for application of our PsXR in constructing a more balanced XR-XDH pathway in recombinant xylose-fermenting S. cerevisiae strains.

Dataset Information

Comparative proteomic analysis of a new adaptive Pichia Stipitis strain to furfural, a lignocellulosic inhibitory compound.

Publications

Comparative proteomic analysis of a new adaptive Pichia Stipitis strain to furfural, a lignocellulosic inhibitory compound.

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