Differential regulation of Pleurotus ostreatus dye peroxidases gene expression in response to dyes and potential application of recombinant Pleos-DyP1 in decolorization.

Dye-decolorizing peroxidase (DyP) from the white rot basidiomycete Pleurotus ostreatus is a heme peroxidase able to oxidize diverse substrates, including recalcitrant phenols and dyes. This study analyzed the effect of chemical dyes on P. ostreatus growth, DyP activity and the expression of four Pleos-dyp genes during the time-course of Pleurotus ostreatus cultures containing either Acetyl Yellow G (AYG), Remazol Brilliant Blue R (RBBR) or Acid Blue 129 (AB129) dyes. Additionally, Pleos DyP1 was heterologously expressed in the filamentous fungus Trichoderma atroviride in order to explore the potential of a secreted recombinant enzyme for decolorizing different dyes in cultures and plate assays. The addition of dyes had an induction effect on the enzymatic activity, with the fermentations undertaken using RBBR and AYG dyes presenting the highest total DyP activity. DyP gene expression profiles displayed up/down regulation during the culture of three Pleos-dyp genes (Pleos-dyp1, Pleos-dyp2 and Pleos-dyp4), while Pleos-dyp3 transcript was not detected under any of the culture conditions studied. A 14-fold relative induction level (log2) increase for Pleos-dyp2 and Pleos-dyp4 in AB129 and AYG, respectively, was also found. The presence of AB129 resulted in the highest Pleos-dyp1 gene induction and repression level, corresponding to 11.83 and -14.6-fold relative expression and repression levels, respectively. The lowest expression level of all genes was observed in RBBR, a response which is associated with the growth phase. The filamentous fungus Trichoderma atroviride was successfully transformed for the heterologous expression of Pleos-dyp1. The modified strains (TaDyP) were able to decolorize mono-azo, di-azo, anthraquinone and anthracenedione dyes with extracellular DyP1 activity found in the culture supernatant. After 96 h of culture, the recombinant TaDyP strains were able to degrade (decolorize) 77 and 34% of 0.05mM AB129 and 0.25mM AYG, respectively.

Enhanced responsiveness and sensitivity to blue light by blr-2 overexpression in Trichoderma atroviride.

Light is an environmental factor that regulates pivotal processes in living organisms, and appropriate perception is key to adaptation to the environment. Blue light activates asexual reproduction in Trichoderma atroviride through transcription factors BLR-1 and BLR-2 which regulate light-responsive genes. Here, we show that blr-2 expression is a limiting factor for photo-perception and photo-transduction. Overexpression of blr-2 resulted in increased photoconidiation and stronger expression of light-induced genes. In contrast, overexpression of blr-1 resulted in reduced photoconidiaton and weaker expression of light-induced genes. blr-2 overexpression caused a marked reduction of growth when the fungus was grown under defined photoperiods, including a period of strong sensitivity to light, followed by a period of insensitivity. Long periods of incubation under this condition permitted recovery of a rhythmic growth similar to that of the wild-type. In addition, blr-2 expression is apparently regulated at the post-transcriptional level through the BLR proteins and its expression level is BLR-1-dependent even in the dark. Finally, we demonstrated that blr-2 overexpression caused higher sensitivity to blue light and we therefore propose that the preformation of BLR-1/BLR-2 complexes is key to adequate light perception in T. atroviride.

Novel light-regulated genes in Trichoderma atroviride: a dissection by cDNA microarrays.

The influence of light on living organisms is critical, not only because of its importance as the main source of energy for the biosphere, but also due to its capacity to induce changes in the behaviour and morphology of nearly all forms of life. The common soil fungus Trichoderma atroviride responds to blue light in a synchronized manner, in time and space, by forming a ring of green conidia at what had been the colony perimeter at the time of exposure (photoconidiation). A putative complex formed by the BLR-1 and BLR-2 proteins in T. atroviride appears to play an essential role as a sensor and transcriptional regulator in photoconidiation. Expression analyses using microarrays containing 1438 unigenes were carried out in order to identify early light response genes. It was found that 2.8 % of the genes were light responsive: 2 % induced and 0.8 % repressed. Expression analysis in blr deletion mutants allowed the demonstration of the occurrence of two types of light responses, a blr-independent response in addition to the expected blr-dependent one, as well as a new role of the BLR proteins in repression of transcription. Exposure of T. atroviride to continuous light helped to establish that the light-responsive genes are subject to photoadaptation. Finally, evidence is provided of red-light-regulated gene expression and a possible crosstalk between the blue and red light signalling pathways.