Transcriptional and enzymatic profiling of Pleurotus ostreatus laccase genes in submerged and solid-state fermentation cultures

Abstract

The genome of the white rot basidiomycete Pleurotus ostreatus includes 12 phenol oxidase (laccase) genes. In this study, we examined their expression profiles in different fungal strains under different culture conditions (submerged and solid cultures) and in the presence of a wheat straw extract, which was used as an inducer of the laccase gene family. We used a reverse transcription-quantitative PCR (RT-qPCR)-based approach and focused on determining the reaction parameters (in particular, the reference gene set for the normalization and reaction efficiency determinations) used to achieve an accurate estimation of the relative gene expression values. The results suggested that (i) laccase gene transcription is upregulated in the induced submerged fermentation (iSmF) cultures but downregulated in the solid fermentation (SSF) cultures, (ii) the Lacc2 and Lacc10 genes are the main sources of laccase activity in the iSmF cultures upon induction with water-soluble wheat straw extracts, and (iii) an additional, as-yet-uncharacterized activity (Unk1) is specifically induced in SSF cultures that complements the activity of Lacc2 and Lacc10. Moreover, both the enzymatic laccase activities and the Lacc gene family transcription profiles greatly differ between closely related strains. These differences can be targeted for biotechnological breeding programs for enzyme production in submerged fermentation reactors.

Fig 1

Relative expression ratios of 10 Lacc genes of P. ostreatus cultured in iSmF (black bars) and on SSF (striped bars) media relative to the expression in the SmF control culture (white bars), according to equation 3 in the text. The bars represent the standard errors of the means of three independent measurements of the same pooled samples (see the text). The statistical significance of the differences between sample means is indicated with open circles (P < 0.05) or solid circles (P < 0.01).

Fig 2

Extracellular laccase activity determined using DMP (A) or biomass (B) as the substrate in SmF (white bars) and iSmF (black bars) cultures. Bars represent the standard deviations of the means of the results of three biological replicate experiments. The statistical significance of the differences between sample means is indicated with open circles (P < 0.05) or solid circles (P < 0.01).

Fig 3

Laccase activity extractable from SSF cultures harvested at different times. The monokaryotic PC15 strain did not show activity during any of the experiments and is not included here. The enzymatic activity was determined using DMP as the substrate. The bars represent the standard deviations of the means of the results of three biological replicate experiments.

Fig 4

Zymograms of the P. ostreatus laccases detected in SmF, iSmF, and SSF cultures. A 37.5-μl volume of protein extract was loaded in each lane.