Molecular and catalytic properties of fungal extracellular cellobiose dehydrogenase produced in prokaryotic and eukaryotic expression systems

Ma, Su; Preims, Marita; Piumi, François; Kappel, Lisa; Schink, Bernhard; Record, Éric; Kracher, Daniel; Ludwig, Roland

doi:10.1186/s12934-017-0653-5

Cited by 38 publications

(24 citation statements)

References 46 publications

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“…This result is similar to the FAD occupancy published for T. reesei-produced CDH from C. thermophilus (CtCDH) [20]. In contrast, CtCDH expressed in P. pastoris contained only 30% FAD in the active site, whereas for native CtCDH 92% occupancy were reported [31].…”

Section: Spectroscopic Analysis (Uv/vis and Fad Loading)supporting

confidence: 87%

“…In contrast, the prokaryotic expression host Escherichia coli would not introduce Oand N-glycans, but successful production of CDH has not been accomplished so far. Only expression of the sole dehydrogenase domain was achieved in E. coli [19,20]. Recombinant production of ascomycetous Corynascus thermophilus CDH in T. reesei and Aspergillus niger [20] indicated that fungal expression hosts could result in a lesser and more uniform glycosylation of recombinant CDH.…”

Section: Introductionmentioning

confidence: 99%

“…Only expression of the sole dehydrogenase domain was achieved in E. coli [19,20]. Recombinant production of ascomycetous Corynascus thermophilus CDH in T. reesei and Aspergillus niger [20] indicated that fungal expression hosts could result in a lesser and more uniform glycosylation of recombinant CDH. Trichoderma reesei is a well-established production host for recombinant protein expression.…”

Section: Introductionmentioning

confidence: 99%

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Heterologous Expression of Phanerochaete Chrysosporium Cellobiose Dehydrogenase in Trichoderma Reesei

Wohlschlager

Csarman

Chang

et al. 2020

Preprint

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Background: Cellobiose 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 basidiomycetous 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). Results: PcCDHTr 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-b-d-glucosaminidase in the supernatant.Conclusions: Heterologous 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.

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Section: Spectroscopic Analysis (Uv/vis and Fad Loading)supporting

confidence: 87%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Heterologous Expression of Phanerochaete Chrysosporium Cellobiose Dehydrogenase in Trichoderma Reesei

Wohlschlager

Csarman

Chang

et al. 2020

Preprint

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“…Although the protein concentration of secreted CDH reported here (1489.30 mg L −1 ) is an improvement on previous attempts at methanol-induced expression, the volumetric activity and specific activity is lower than previously reported [21,[25][26][27]. Lowered specific activity has been observed in recombinant CDH production by P. pastoris due to a sub-stoichiometric occupation of catalytic sites within the flavin adenine dinucleotide (FAD) cofactor, as well as hyper-glycosylation [28]. Further, hyper-glycosylation may have affected the specific activity of both target enzymes, since the reported protein sizes ( Fig.…”

Section: Glycerol Fed-batch Fermentation Kinetics Yields and Productcontrasting

confidence: 61%

Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris

Conacher

García-Aparicio

Coetzee

et al. 2019

Preprint

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Objective: Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. Results: The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489-2780 mg.L -1 ), and were secreted at moderate to high percentages of the total extracellular protein (51-94 %). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5-6-fold, reaching enzyme activity levels (1020-202 U.L -1 ) that could allow their direct application.

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“…The reason can be minor differences in the FAD occupation of the active-site. 23 The observed differences of the IET between DH and CYT measured with the cytochrome c assay (1.27 to 1.98 U mg -1 ) vary to the same extent as the catalytic turnover and indicate that the introduced mutations exert no effect on the IET and CYT mobility.…”

Section: Recombinant Production Of Cdh Variantsmentioning

confidence: 93%

Direct Electron-Transfer Anisotropy of a Site-Specifically Immobilized Cellobiose Dehydrogenase

Ma¹,

Laurent²,

Meneghello

et al. 2019

ACS Catal.

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To study the direct electron transfer (DET) of the multi-cofactor enzyme cellobiose dehydrogenase (CDH) in regard to its orientation on an electrode surface, a recently published, maleimide-based immobilization method was used in combination with sitedirected mutagenesis to establish different orientations on an electrode surface. CDH from Myriococcum thermophilum was chosen for this study, because its protein structure is resolved and the factors influencing the movement of its mobile cytochrome domain (CYT) are established. Seven CDH variants with a surface exposed cysteine residue in different spatial positions were generated for site-specific maleimide coupling. Surface plasmon resonance and cyclic voltammetry showed that all CDH variants, but not the wild-type CDH, bound covalently to gold electrodes or glassy carbon electrodes and were catalytically active. For DET, the CYT domain needs to move from the closed-state conformation, where it obtains an electron from the catalytic FAD cofactor to the open state where it can donate an electron to the electrode. We therefore hypothesized that the mobility of the CYT domain and its distance to the electrode is central for DET. We found that the uniform spatial orientations of CDH influenced DET as follows: an orientation of the two-domain enzyme on the side, with CYT in proximity to the electrode resulted in high DET currents. Orientations with a bigger distance between CYT and the electrode, or orientations where CYT could not swing back to the dehydrogenase domain (DH) to form the closed enzyme conformation, reduced DET. In the latter case calcium ions, that stabilize the closed conformation of CDH, fully recovered DET. The study demonstrates, that a mobile CYT domain can compensate unfavorable orientations of the catalytic domain to a great extent and allows CDH as a multi-cofactor enzyme to transfer electrons even in awkward orientations. The mobile CYT domain reduces the anisotropy of DET, which is also essential for CDH's physiological function as an extracellular, electron transferring enzyme.

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Molecular and catalytic properties of fungal extracellular cellobiose dehydrogenase produced in prokaryotic and eukaryotic expression systems

Cited by 38 publications

References 46 publications

Heterologous Expression of Phanerochaete Chrysosporium Cellobiose Dehydrogenase in Trichoderma Reesei

Heterologous Expression of Phanerochaete Chrysosporium Cellobiose Dehydrogenase in Trichoderma Reesei

Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris

Direct Electron-Transfer Anisotropy of a Site-Specifically Immobilized Cellobiose Dehydrogenase

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