Regulation of dye-decolorizing peroxidase gene expression in Pleurotus ostreatus grown on glycerol as the carbon source

Abstract

Dye-decolorizing peroxidases (DyPs) (E.C. 1.11.1.19) are heme peroxidases that catalyze oxygen transfer reactions similarly to oxygenases. DyPs utilize hydrogen peroxide (H2O2) both as an electron acceptor co-substrate and as an electron donor when oxidized to their respective radicals. The production of both DyPs and lignin-modifying enzymes (LMEs) is regulated by the carbon source, although less readily metabolizable carbon sources do improve LME production. The present study analyzed the effect of glycerol on Pleurotus ostreatus growth, total DyP activity, and the expression of three Pleos-dyp genes (Pleos-dyp1, Pleos-dyp2 and Pleos-dyp4), via real-time RT-qPCR, monitoring the time course of P. ostreatus cultures supplemented with either glycerol or glucose and Acetyl Yellow G (AYG) dye. The results obtained indicate that glycerol negatively affects P. ostreatus growth, giving a biomass production of 5.31 and 5.62 g/L with respective growth rates (micra; m) of 0.027 and 0.023 h−1 for fermentations in the absence and presence of AYG dye. In contrast, respective biomass production levels of 7.09 and 7.20 g/L and growth rates (μ) of 0.033 and 0.047 h−1 were observed in equivalent control fermentations conducted with glucose in the absence and presence of AYG dye. Higher DyP activity levels, 4,043 and 4,902 IU/L, were obtained for fermentations conducted on glycerol, equivalent to 2.6-fold and 3.16-fold higher than the activity observed when glucose is used as the carbon source. The differential regulation of the DyP-encoding genes in P. ostreatus were explored, evaluating the carbon source, the growth phase, and the influence of the dye. The global analysis of the expression patterns throughout the fermentation showed the up- and down- regulation of the three Pleos-dyp genes evaluated. The highest induction observed for the control media was that found for the Pleos-dyp1 gene, which is equivalent to an 11.1-fold increase in relative expression (log2) during the stationary phase of the culture (360 h), and for the glucose/AYG media was Pleos-dyp-4 with 8.28-fold increase after 168 h. In addition, glycerol preferentially induced the Pleos-dyp1 and Pleos-dyp2 genes, leading to respective 11.61 and 4.28-fold increases after 144 h. After 360 and 504 h of culture, 12.86 and 4.02-fold increases were observed in the induction levels presented by Pleos-dyp1 and Pleos-dyp2, respectively, in the presence of AYG. When transcription levels were referred to those found in the control media, adding AYG led to up-regulation of the three dyp genes throughout the fermentation. Contrary to the fermentation with glycerol, where up- and down-regulation was observed. The present study is the first report describing the effect of a less-metabolizable carbon source, such as glycerol, on the differential expression of DyP-encoding genes and their corresponding activity.