Mutagenesis of Trichoderma reesei endoglucanase I: impact of expression host on activity and stability at elevated temperatures

Chokhawala, Harshal A.; Roche, Christine M.; Kim, Tae‐Wan; Atreya, Meera E.; Vegesna, Neeraja; Dana, Craig M.; Blanch, Harvey W.; Clark, Douglas S.

doi:10.1186/s12896-015-0118-z

Cited by 61 publications

(41 citation statements)

References 44 publications

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“…The secretion yield of rh Tr EG II obtained in this study was comparable to that reported in the literature [26, 29]. However, the amount of secreted rh Tr EG I was higher than the previously reported value (5 mg/L).…”

Section: Resultssupporting

confidence: 86%

Conferring cellulose-degrading ability to Yarrowia lipolytica to facilitate a consolidated bioprocessing approach

Guo

Duquesne

Bozonnet

et al. 2017

Biotechnol Biofuels

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Background Yarrowia lipolytica, one of the most widely studied “nonconventional” oleaginous yeast species, is unable to grow on cellulose. Recently, we identified and overexpressed two endogenous β-glucosidases in Y. lipolytica, thus enabling this yeast to use cello-oligosaccharides as a carbon source for growth. Using this engineered yeast platform, we have now gone further toward building a fully cellulolytic Y. lipolytica for use in consolidated bioprocessing of cellulose.ResultsInitially, different essential enzyme components of a cellulase cocktail (i.e,. cellobiohydrolases and endoglucanases) were individually expressed in Y. lipolytica in order to ascertain the viability of the strategy. Accordingly, the Trichoderma reesei endoglucanase I (TrEG I) and II (TrEG II) were secreted as active proteins in Y. lipolytica, with the secretion yield of EG II being twice that of EG I. Characterization of the purified His-tagged recombinant EG proteins (rhTrEGs) revealed that rhTrEG I displayed higher specific activity than rhTrEG II on both cellotriose and insoluble cellulosic substrates, such as Avicel, β-1, 3 glucan, β-1, 4 glucan, and PASC. Similarly, cellobiohydrolases, such as T. reesei CBH I and II (TrCBH I and II), and the CBH I from Neurospora crassa (NcCBH I) were successfully expressed in Y. lipolytica. However, the yield of the expressed TrCBH I was low, so work on this was not pursued. Contrastingly, rhNcCBH I was not only well expressed, but also highly active on PASC and more active on Avicel (0.11 U/mg) than wild-type TrCBH I (0.065 U/mg). Therefore, work was pursued using a combination of NcCBH I and TrCBH II. The quantification of enzyme levels in culture supernatants revealed that the use of a hybrid promoter instead of the primarily used TEF promoter procured four and eight times more NcCBH I and TrCBH II expressions, respectively. Finally, the coexpression of the previously described Y. lipolytica β-glucosidases, the CBH II, and EG I and II from T. reesei, and the N. crassa CBH I procured an engineered Y. lipolytica strain that was able to grow both on model cellulose substrates, such as highly crystalline Avicel, and on industrial cellulose pulp, such as that obtained using an organosolv process.ConclusionsA Y. lipolytica strain coexpressing six cellulolytic enzyme components has been successfully developed. In addition, the results presented show how the recombinant strain can be optimized, for example, using artificial promoters to tailor expression levels. Most significantly, this study has provided a demonstration of how the strain can grow on a sample of industrial cellulose as sole carbon source, thus revealing the feasibility of Yarrowia-based consolidated bioprocess for the production of fuel and chemical precursors. Further, enzyme and strain optimization, coupled to appropriate process design, will undoubtedly lead to much better performances in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0819-8) contains supplementary material, ...

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Section: Resultssupporting

confidence: 86%

Conferring cellulose-degrading ability to Yarrowia lipolytica to facilitate a consolidated bioprocessing approach

Guo

Duquesne

Bozonnet

et al. 2017

Biotechnol Biofuels

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“…With a 65% residual activity after more than 15 days (365 h) at T opt , the half-life of BsGH7-3 is amongst the highest reported compared to other GH7 endoglucanases, particularly at high pH [58]. Chokhawala et al reported the expression of an engineered T. reesei EGI variant in T. reesei (G230A/D113S/D115T Tr_TrEG1) with a half-life of 161 h at 60 °C and pH 4.85 in comparison to the recombinant ( T. reesei host) wild-type TrEG1 with a half-life of 74 h at 60 °C, pH 4.85 [62]. Another EG from Trichoderma harzianum has also been reported to be very stable, with a little change in activity after 2 months of incubation (Additional file 1: Table S3).…”

Section: Discussionmentioning

confidence: 99%

Genome-wide characterization of cellulases from the hemi-biotrophic plant pathogen, Bipolaris sorokiniana, reveals the presence of a highly stable GH7 endoglucanase

Aich

Singh

Kundu

et al. 2017

Biotechnol Biofuels

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Background Bipolaris sorokiniana is a filamentous fungus that causes spot blotch disease in cereals like wheat and has severe economic consequences. However, information on the identities and role of the cell wall-degrading enzymes (CWDE) in B. sorokiniana is very limited. Several fungi produce CWDE like glycosyl hydrolases (GHs) that help in host cell invasion. To understand the role of these CWDE in B. sorokiniana, the first step is to identify and annotate all possible genes of the GH families like GH3, GH6, GH7, GH45 and AA9 and then characterize them biochemically.ResultsWe confirmed and annotated the homologs of GH3, GH6, GH7, GH45 and AA9 enzymes in the B. sorokiniana genome using the sequence and domain features of these families. Quantitative real-time PCR analyses of these homologs revealed that the transcripts of the BsGH7-3 (3rd homolog of the GH 7 family in B. sorokiniana) were most abundant. BsGH7-3, the gene of BsGH7-3, was thus cloned into pPICZαC Pichia pastoris vector and expressed in X33 P. pastoris host to be characterized. BsGH7-3 enzyme showed a temperature optimum of 60 °C and a pHopt of 8.1. BsGH7-3 was identified to be an endoglucanase based on its broad substrate specificity and structural comparisons with other such endoglucanases. BsGH7-3 has a very long half-life and retains 100% activity even in the presence of 4 M NaCl, 4 M KCl and 20% (v/v) ionic liquids. The enzyme activity is stimulated up to fivefold in the presence of Mn+2 and Fe+2 without any deleterious effects on enzyme thermostability.ConclusionsHere we reanalysed the B. sorokiniana genome and selected one GH7 enzyme for further characterization. The present work demonstrates that BsGH7-3 is an endoglucanase with a long half-life and no loss in activity in the presence of denaturants like salt and ionic liquids, and lays the foundation towards exploring the Bipolaris genome for other cell wall-degrading enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0822-0) contains supplementary material, which is available to authorized users.

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“…Previous studies describing pGlu function in CBH-1 did not verify the presence or absence of the N-terminal pGlu modification (12, 13). To address this question directly, we conducted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of trypsin-digested CBH-1 purified from WT cells versus that from the Δ qc - 1 Δ qc - 2 mutant; the N-terminal CBH-1 peptide resulting from trypsin digestion is QAVCSLTAETHPSLNWSK.…”

Section: Resultsmentioning

confidence: 84%

“…TeCBH-1 and TeEG1 heterologously expressed in S. cerevisiae show reduced thermal stability and presumably lack the N-terminal pGlu modification (12, 13). In hindsight, this result is surprising because a QC homolog is present in the S. cerevisiae genome (which is uncharacterized, but a GFP-tagged version is reported to localize to the ER; YFR018C) (41) (see Fig.…”

Section: Discussionmentioning

confidence: 99%

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Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes

Dana

Iavarone

et al. 2017

mBio

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The breakdown of plant biomass to simple sugars is essential for the production of second-generation biofuels and high-value bioproducts. Currently, enzymes produced from filamentous fungi are used for deconstructing plant cell wall polysaccharides into fermentable sugars for biorefinery applications. A post-translational N-terminal pyroglutamate modification observed in some of these enzymes occurs when N-terminal glutamine or glutamate is cyclized to form a five-membered ring. This modification has been shown to confer resistance to thermal denaturation for CBH-1 and EG-1 cellulases. In mammalian cells, the formation of pyroglutamate is catalyzed by glutaminyl cyclases. Using the model filamentous fungus Neurospora crassa, we identified two genes (qc-1 and qc-2) that encode proteins homologous to mammalian glutaminyl cyclases. We show that qc-1 and qc-2 are essential for catalyzing the formation of an N-terminal pyroglutamate on CBH-1 and GH5-1. CBH-1 and GH5-1 produced in a Δqc-1 Δqc-2 mutant, and thus lacking the N-terminal pyroglutamate modification, showed greater sensitivity to thermal denaturation, and for GH5-1, susceptibility to proteolytic cleavage. QC-1 and QC-2 are endoplasmic reticulum (ER)-localized proteins. The pyroglutamate modification is predicted to occur in a number of additional fungal proteins that have diverse functions. The identification of glutaminyl cyclases in fungi may have implications for production of lignocellulolytic enzymes, heterologous expression, and biotechnological applications revolving around protein stability.

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Mutagenesis of Trichoderma reesei endoglucanase I: impact of expression host on activity and stability at elevated temperatures

Cited by 61 publications

References 44 publications

Conferring cellulose-degrading ability to Yarrowia lipolytica to facilitate a consolidated bioprocessing approach

Conferring cellulose-degrading ability to Yarrowia lipolytica to facilitate a consolidated bioprocessing approach

Genome-wide characterization of cellulases from the hemi-biotrophic plant pathogen, Bipolaris sorokiniana, reveals the presence of a highly stable GH7 endoglucanase

Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes

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