Improvement of ligninolytic properties by recombinant expression of glyoxal oxidase gene in hyper lignin-degrading fungus Phanerochaete sordida YK-624

Abstract

Glyoxal oxidase (GLOX) is a source of the extracellular H2O2 required for the oxidation reactions catalyzed by the ligninolytic peroxidases. In the present study, the GLOX-encoding gene (glx) of Phanerochaete chrysosporium was cloned, and bee2 promoter of P. sordida YK-624 was used to drive the expression of glx. The expression plasmid was transformed into a P. sordida YK-624 uracil auxotrophic mutant (strain UV-64), and 16 clones were obtained as GLOX-introducing transformants. These transformants showed higher GLOX activities than wild-type P. sordida YK-624 and control transformants harboring marker plasmid. RT-PCR analysis indicated that the increased GLOX activity was associated with elevated recombinant glx expression. Moreover, these transformants showed higher ligninolytic activity than control transformants. These results suggest that the ligninolytic properties of white-rot fungi can be improved by recombinant expression of glx.

Lignin degradation: microorganisms, enzymes involved, genomes analysis and evolution

“…Glyoxal oxidase generates extracellular hydrogen peroxide by the oxidation of a variety of simple dicarbonyl and hydroxycarbonyl compounds, especially glyoxal and methylglyoxal (Kersten and Cullen 1993 ; Yamada et al. 2014 ). Based on an unusual free radical-coupled copper active site, GLOX was classified in the CAZy family among other copper radical oxidases in the family AA5 (Whittaker et al.…”

Structure–function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family

“…GenBank identifiers (UniProt identifier Q01745 in the case of the F. graminarium GalOx) are given for all sequences available in the public release of the CAZy Database 16 as of June 2015; full genus and species names of source organisms can be obtained from the respective GenBank entries. AA5 members for which enzymological data have been conclusively linked to protein sequence are indicated as (methyl)glyoxal oxidases (GlyoxOx) 9 13 14 17 18 , galactose oxidases (GalOx; refs 19 , 27 , 34 , 35 , 68 and references therein), or general alcohol oxidases (AlcOx; this study).…”

“…Strikingly, the recent CAZy classification of AA5 reveals a paucity of sequence-correlated functional and structural information in this family. Indeed, since the initial discovery nearly 30 years ago of (methyl)glyoxal oxidase activity in the white-rot basidiomycete Phanerochaete chrysosporium 8 , only the archetype Pch Glx1 (refs 13 , 14 , 17 ), a strain-specific point variant 18 , and a homologue from the phytopathogenic fungus Ustilago maydis 9 have been recombinantly produced and enzymatically characterized, while no three-dimensional (3D) structures have been solved for any AA5_1 members 8 . Studies on AA5_2 members have essentially exclusively focused on the archetypal galactose 6-oxidase from the cereal head blight ascomycete Fusarium graminearum (anamorph of Gibberella zeae ), encompassing recombinant production, biochemical and kinetic analyses, spectroscopy, crystallography, enzyme engineering and applications 5 10 .…”

Characterization of a New Glyoxal Oxidase from the Thermophilic Fungus Myceliophthora thermophila M77: Hydrogen Peroxide Production Retained in 5-Hydroxymethylfurfural Oxidation

“…Another reported substrate that is oxidized by GLOx is glycerol, formed in bulk during biodiesel production [25]. Experimental evidence suggests that GLOxes need to be activated by other enzymes such as lignin peroxidases and can enhance lignolytic activity of white-rot fungi in biomass degradation [26,29,30]. Glycoaldehydes, products from lignin degradation and inhibitors of bioethanol fermentation, have been suggested to be GLOx substrates [31].…”