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Molecular characterization and heterologous expression of the gene encoding a low-molecular-mass endoglucanase from Trichoderma reesei QM9414.

ABSTRACT: We have isolated the genomic and cDNA clones encoding EG III (a low-molecular-mass endo-beta-1,4-glucanase) gene from Trichoderma reesei QM9414. The nucleotide sequence of the cDNA fragment was verified to contain a 702-bp open reading frame that encodes a 234-amino-acid propeptide. The deduced protein sequence has significant homologies with family H endo-beta-1,4-glucanases. The 16-amino-acid N-terminal sequence was shown to function as a leader peptide for possible secretion. Northern blot analysis showed that the EG III gene transcript, with a length of about 700 bp, was expressed markedly by cellulose but not by glucose. The protein has been expressed as a mature form in Escherichia coli and as secreted forms in Saccharomyces cerevisiae and Schizosaccharomyces pombe under the control of tac, alcohol dehydrogenase (ADH1), and human cytomegalovirus promoters, respectively. The S. cerevisiae and Schizosaccharomyces pombe recombinant strains showed strong cellulolytic activities on agar plates containing carboxymethyl cellulose. The E. coli strain expressed small amounts of EG III in an active form and large amounts of EG III in an inactive form. The molecular masses of the recombinant EG IIIs were estimated to be 25, 28, and 29 kDa for E. coli, S. cerevisiae, and Schizosaccharomyces pombe, respectively, by immunoblot analysis following sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. Parts of the yeast recombinant EG IIIs decreased their molecular masses to 25 kDa after treatment with endoglycosidase H and alpha-mannosidase, suggesting that they are N glycosylated at least partly.

SUBMITTER: Okada H

PROVIDER: S-EPMC106082 | biostudies-literature | 1998 Feb

REPOSITORIES: biostudies-literature

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Molecular characterization and heterologous expression of the gene encoding a low-molecular-mass endoglucanase from Trichoderma reesei QM9414.

Okada H H Tada K K Sekiya T T Yokoyama K K Takahashi A A Tohda H H Kumagai H H Morikawa Y Y

Applied and environmental microbiology 19980201 2

We have isolated the genomic and cDNA clones encoding EG III (a low-molecular-mass endo-beta-1,4-glucanase) gene from Trichoderma reesei QM9414. The nucleotide sequence of the cDNA fragment was verified to contain a 702-bp open reading frame that encodes a 234-amino-acid propeptide. The deduced protein sequence has significant homologies with family H endo-beta-1,4-glucanases. The 16-amino-acid N-terminal sequence was shown to function as a leader peptide for possible secretion. Northern blot an ...[more]

PMID: 9464393

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Project description:BackgroundCellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDHTr) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDHPp).ResultsPcCDHTr was recombinantly produced with a yield of 600 U L-1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDHTr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDHTr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDHTr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-D-glucosamine additions at the six potential N-glycosylation sites of PcCDHTr, which indicates the presence of an endo-N-acetyl-?-D-glucosaminidase in the supernatant.ConclusionsHeterologous production of PcCDHTr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDHTr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDHTr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells.

Project description:BackgroundTrichoderma reesei is a key cellulase source for economically saccharifying cellulosic biomass for the production of biofuels. Lignocellulose hydrolysis at temperatures above the optimum temperature of T. reesei cellulases (~50°C) could provide many significant advantages, including reduced viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature biomass pretreatment, and faster rates of hydrolysis. These potential advantages motivate efforts to engineer T. reesei cellulases that can hydrolyze lignocellulose at temperatures ranging from 60-70°C.ResultsA B-factor guided approach for improving thermostability was used to engineer variants of endoglucanase I (Cel7B) from T. reesei (TrEGI) that are able to hydrolyze cellulosic substrates more rapidly than the recombinant wild-type TrEGI at temperatures ranging from 50-70°C. When expressed in T. reesei, TrEGI variant G230A/D113S/D115T (G230A/D113S/D115T Tr_TrEGI) had a higher apparent melting temperature (3°C increase in Tm) and improved half-life at 60°C (t1/2 = 161 hr) than the recombinant (T. reesei host) wild-type TrEGI (t1/2 = 74 hr at 60°C, Tr_TrEGI). Furthermore, G230A/D113S/D115T Tr_TrEGI showed 2-fold improved activity compared to Tr_TrEGI at 65°C on solid cellulosic substrates, and was as efficient in hydrolyzing cellulose at 60°C as Tr_TrEGI was at 50°C. The activities and stabilities of the recombinant TrEGI enzymes followed similar trends but differed significantly in magnitude depending on the expression host (Escherichia coli cell-free, Saccharomyces cerevisiae, Neurospora crassa, or T. reesei). Compared to N.crassa-expressed TrEGI, S. cerevisiae-expressed TrEGI showed inferior activity and stability, which was attributed to the lack of cyclization of the N-terminal glutamine in Sc_TrEGI and not to differences in glycosylation. N-terminal pyroglutamate formation in TrEGI expressed in S. cerevisiae was found to be essential in elevating its activity and stability to levels similar to the T. reesei or N. crassa-expressed enzyme, highlighting the importance of this ubiquitous modification in GH7 enzymes.ConclusionStructure-guided evolution of T. reesei EGI was used to engineer enzymes with increased thermal stability and activity on solid cellulosic substrates. Production of TrEGI enzymes in four hosts highlighted the impact of the expression host and the role of N-terminal pyroglutamate formation on the activity and stability of TrEGI enzymes.

Dataset Information

Molecular characterization and heterologous expression of the gene encoding a low-molecular-mass endoglucanase from Trichoderma reesei QM9414.

Publications

Molecular characterization and heterologous expression of the gene encoding a low-molecular-mass endoglucanase from Trichoderma reesei QM9414.

Similar Datasets

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