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Phanerochaete chrysosporium glyoxal oxidase is encoded by two allelic variants: structure, genomic organization, and heterologous expression of glx1 and glx2.

ABSTRACT: A cDNA clone (glx-2c) encoding glyoxal oxidase (GLOX) was isolated from a Phanerochaete chrysosporium lambda gt11 library, and its nucleotide sequence was shown to be distinct from that of the previously described clone glx-1c (P. J. Kersten and D. Cullen, Proc. Natl. Acad. Sci. USA 90:7411-7413, 1993). Genomic clones corresponding to both cDNAs were also isolated and sequenced. overall nucleotide sequence identity was 98%, and the predicted proteins differed by a single residue: Lys-308<==>Thr-308. Analyses of parental dikaryotic strain BKM-F-1767 and homokaryotic progeny firmly established allelism for these structural variants. Southern blots of pulsed-field gels localized the GLOX gene (glx) to a dimorphic chromosome separate from the peroxidase and cellobiohydrolase genes of P. chrysosporium. Controlled expression of active GLOX was obtained from Aspergillus nidulans transformants when glx-1c was fused to the promoter and secretion signal of the A. niger glucoamylase gene. The GLOX isozyme corresponding to glx-2c was also efficiently secreted by A. nidulans following site-specific mutagenesis of the expression vector at codon 308 of glx-1c.

SUBMITTER: Kersten PJ

PROVIDER: S-EPMC177449 | biostudies-other | 1995 Nov

REPOSITORIES: biostudies-other

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Phanerochaete chrysosporium glyoxal oxidase is encoded by two allelic variants: structure, genomic organization, and heterologous expression of glx1 and glx2.

Kersten P J PJ Witek C C vanden Wymelenberg A A Cullen D D

Journal of bacteriology 19951101 21

A cDNA clone (glx-2c) encoding glyoxal oxidase (GLOX) was isolated from a Phanerochaete chrysosporium lambda gt11 library, and its nucleotide sequence was shown to be distinct from that of the previously described clone glx-1c (P. J. Kersten and D. Cullen, Proc. Natl. Acad. Sci. USA 90:7411-7413, 1993). Genomic clones corresponding to both cDNAs were also isolated and sequenced. overall nucleotide sequence identity was 98%, and the predicted proteins differed by a single residue: Lys-308<==>Thr- ...[more]

PMID: 7592374

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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-β-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.

Dataset Information

Phanerochaete chrysosporium glyoxal oxidase is encoded by two allelic variants: structure, genomic organization, and heterologous expression of glx1 and glx2.

Publications

Phanerochaete chrysosporium glyoxal oxidase is encoded by two allelic variants: structure, genomic organization, and heterologous expression of glx1 and glx2.

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