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Design, expression and functional characterization of a thermostable xylanase from Trichoderma reesei.

ABSTRACT: Xylanases isolated from microorganisms such as the Trichoderma reesei have attracted considerable research interest because of their potential in various industrial applications. However, naturally isolated xylanases cannot withstand harsh conditions such as high temperature and basic pH. In this study, we performed structural analysis of the major T. reesei xylanase (Xyn2), and novel flexible regions of the enzyme were identified based on B-factor, a molecular dynamics (MD) parameter. To improve thermostability of the Xyn2, disulfide bonds were introduced into the unstable flexible region by using site-directed mutagenesis and two recombinant xylanases, XM1 (Xyn2Cys12-52) and XM2 (Xyn2Cys59-149) were successfully expressed in Pichia pastoris. Secreted recombinant Xyn2 was estimated by SDS-PAGE to be 24 kDa. Interestingly, the half-lives of XM1 and XM2 at 60°C were 2.5- and 1.8- fold higher, respectively than those of native Xyn2. The XM1 also exhibited improved pH stability and maintained more than 60% activity over pH values ranging from 2.0 to 10.0. However, the specific activity and catalytic efficiency of XM1 was decreased as compared to those of XM2 and native Xyn2. Our results will assist not only in elucidating of the interactions between protein structure and function, but also in rational target selection for improving the thermostability of enzymes.

SUBMITTER: He J

PROVIDER: S-EPMC6334952 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Design, expression and functional characterization of a thermostable xylanase from Trichoderma reesei.

He Jun J Tang Feng F Chen Daiwen D Yu Bing B Luo Yuheng Y Zheng Ping P Mao Xiangbing X Yu Jie J Yu Feng F

PloS one 20190116 1

Xylanases isolated from microorganisms such as the Trichoderma reesei have attracted considerable research interest because of their potential in various industrial applications. However, naturally isolated xylanases cannot withstand harsh conditions such as high temperature and basic pH. In this study, we performed structural analysis of the major T. reesei xylanase (Xyn2), and novel flexible regions of the enzyme were identified based on B-factor, a molecular dynamics (MD) parameter. To improv ...[more]

PMID: 30650138

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Project description:BackgroundLignocellulose biomass has been investigated as a feedstock for second generation biofuels and other value-added products. Some of the processes for biofuel production utilize cellulases and hemicellulases to convert the lignocellulosic biomass into a range of soluble sugars before fermentation with microorganisms such as yeast Saccharomyces cerevisiae. One of these sugars is L-arabinose, which cannot be utilized naturally by yeast. The first step in L-arabinose catabolism is its transport into the cells, and yeast lacks a specific transporter, which could perform this task.ResultsWe identified Trire2_104072 of Trichoderma reesei as a potential L-arabinose transporter based on its expression profile. This transporter was described already in 2007 as D-xylose transporter XLT1. Electrophysiology experiments with Xenopus laevis oocytes and heterologous expression in yeast revealed that Trire2_104072 is a high-affinity L-arabinose symporter with a Km value in the range of [Formula: see text] 0.1-0.2 mM. It can also transport D-xylose but with low affinity (Km [Formula: see text] 9 mM). In yeast, L-arabinose transport was inhibited slightly by D-xylose but not by D-glucose in an assay with fivefold excess of the inhibiting sugar. Comparison with known L-arabinose transporters revealed that the expression of Trire2_104072 enabled yeast to uptake L-arabinose at the highest rate in conditions with low extracellular L-arabinose concentration. Despite the high specificity of Trire2_104072 for L-arabinose, the growth of its T. reesei deletion mutant was only affected at low L-arabinose concentrations.ConclusionsDue to its high affinity for L-arabinose and low inhibition by D-glucose or D-xylose, Trire2_104072 could serve as a good candidate for improving the existing pentose-utilizing yeast strains. The discovery of a highly specific L-arabinose transporter also adds to our knowledge of the primary metabolism of T. reesei. The phenotype of the deletion strain suggests the involvement of other transporters in L-arabinose transport in this species.

Project description:Trichoderma reesei is known for its ability to produce large amounts of extracellular proteins and is one of the most important industrially used filamentous fungus. Xylanase regulator 1 (XYR1) as the master regulator is responsible for the activation of cellulase and hemicellulase gene expression, normally under inducing conditions. It has been reported that strains with point mutations in certain areas of xyr1 bypass the carbon catabolite repression, allowing cellulase and hemicellulase production even in the presence of glucose. These mutations also change the profile of produced proteins, shifting it more towards xylanase production, and increase the overall protein production in inducing conditions. However, how these mutations alter the metabolism and other cellular processes to cause these changes remains unclear. In the present study, our aim was to explore changes caused by a point mutation in xyr1 on transcriptomic and metabolic level to better understand the reasons behind the increased protein production in both repressing glucose and inducing lactose conditions. We observed that the xyr1 mutant strain built more biomass and produced more extracellular proteins during growth on lactose compared to the wild type xyr1 strain. Genes involved in oxidoreductive D-galactose catabolism pathway were upregulated in the xyr1 mutant strain, potentially contributing to the more efficient utilization of lactose. The shift from utilizing lactose to using glucose as carbon source seemed faster in the xyr1 mutant strain. Clustering and enrichment analysis showed over-representation of mitochondria-related Gene Ontology terms in clusters where gene expression was higher in the xyr1 mutant, indicating that mitochondria play a role in the altered metabolic state associated with the xyr1 mutation. Metabolomics revealed that free tyrosine was more abundant in the xyr1 mutant strain in all measured timepoints, whereas multiple fatty acids were less abundant in the mutant strain on glucose. The results contribute to more in-depth knowledge on T. reesei physiology and aid in finding new targets for improved protein production.

Project description:BackgroundFilamentous fungus Trichoderma reesei has been widely used as a workhorse for cellulase and xylanase productions. Xylanase has been reported as the crucial accessory enzyme in the degradation of lignocellulose for higher accessibility of cellulase. In addition, the efficient hydrolysis of xylan needs the co-work of multiple xylanolytic enzymes, which rise an increasing demand for the high yield of xylanase for efficient biomass degradation.ResultsIn this study, a xylanase hyper-producing system in T. reesei was established by tailoring two transcription factors, XYR1 and ACE1, and homologous overexpression of the major endo-xylanase XYNII. The expressed xylanase cocktail contained 5256 U/mL xylanase activity and 9.25 U/mL β-xylosidase (pNPXase) activity. Meanwhile, the transcription level of the xylanolytic genes in the strain with XYR1 overexpressed was upregulated, which was well correlated with the amount of XYR1-binding sites. In addition, the higher expression of associated xylanolytic enzymes would result in more efficient xylan hydrolysis. Besides, 2310-3085 U/mL of xylanase activities were achieved using soluble carbon source, which was more efficient and economical than the traditional strategy of xylan induction. Unexpectedly, deletion of ace1 in C30OExyr1 did not give any improvement, which might be the result of the disturbed function of the complex formed between ACE1 and XYR1. The enzymatic hydrolysis of alkali pretreated corn stover using the crude xylanase cocktails as accessory enzymes resulted in a 36.64% increase in saccharification efficiency with the ratio of xylanase activity vs FPase activity at 500, compared to that using cellulase alone.ConclusionsAn efficient and economical xylanase hyper-producing platform was developed in T. reesei RUT-C30. The novel platform with outstanding ability for crude xylanase cocktail production would greatly fit in biomass degradation and give a new perspective of further engineering in T. reesei for industrial purposes.

Dataset Information

Design, expression and functional characterization of a thermostable xylanase from Trichoderma reesei.

Publications

Design, expression and functional characterization of a thermostable xylanase from Trichoderma reesei.

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