Research Article: A calmodulin inhibitor, W-7 influences the effect of cyclic adenosine 3′, 5′-monophosphate signaling on ligninolytic enzyme gene expression in Phanerochaete chrysosporium

Date Published: January 24, 2012

Publisher: Springer

Author(s): Takaiku Sakamoto, Yuki Yao, Yoshifumi Hida, Yoichi Honda, Takashi Watanabe, Wataru Hashigaya, Kazumi Suzuki, Toshikazu Irie.


The capacity of white-rot fungi to degrade wood lignin may be highly applicable to the development of novel bioreactor systems, but the mechanisms underlying this function are not yet fully understood. Lignin peroxidase (LiP) and manganese peroxidase (MnP), which are thought to be very important for the ligninolytic property, demonstrated increased activity in Phanerochaete chrysosporium RP-78 (FGSC #9002, ATCC MYA-4764™) cultures following exposure to 5 mM cyclic adenosine 3′, 5′-monophosphate (cAMP) and 500 μM 3′-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that transcription of most LiP and MnP isozyme genes was statistically significantly upregulated in the presence of the cAMP and IBMX compared to the untreated condition. However, 100 μM calmodulin (CaM) inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which had insignificant effects on fungal growth and intracellular cAMP concentration, not only offset the increased activity and transcription induced by the drugs, but also decreased them to below basal levels. Like the isozyme genes, transcription of the CaM gene (cam) was also upregulated by cAMP and IBMX. These results suggest that cAMP signaling functions to increase the transcription of LiP and MnP through the induction of cam transcription.

Partial Text

White-rot fungi are known to have a powerful ligninolytic system that can completely degrade wood lignin (Kirk and Farrell 1987; Kirk et al. 1975) as well as persistent organic pollutants such as dioxin (Bumpus et al. 1985). This ability may be applicable to the construction of a novel potent bioreactor system to convert wood to potent materials and energy sources with low environmental load and to bioremediate polluted environments. However, the ligninolytic property of these fungi is attributable to many known and unknown enzyme genes, expression of which is inductive, and the factors that determine this expression are not completely understood. The lack of knowledge regarding the ligninolytic property of these fungi is an impediment to the development of a highly effective lignin-degrading fungal strain for the construction of an efficient bioreactor system (Cullen and Kersten 2004). The identification of a master regulator that regulates the entire ligninolytic system in white-rot fungi could be used as a target for breeding a high lignin-degrading strain and for furthering our understanding of the lignin-degradation system in these fungi.

Expression of all lip and mnp isozyme genes except lipC, lipF, lipH was statistically significantly increased compared to the control condition with the absence of drugs (Figure 4). This finding strongly suggests that cAMP signaling increases lip and mnp transcription levels. We have also previously reported that CaM transcription was repressed following exposure to atropine (Minami et al. 2009), and that lip and mnp isozyme gene transcripts were downregulated by addition of the CaM inhibitor, W-7 (Sakamoto et al. 2010). These observations indicated that atropine decreased endogenous cAMP concentration, which resulted in insufficient cAMP signaling to induce upregulation of cam gene transcription. This evidence is strongly supported by the observation that cam gene transcription was also increased by the addition of cAMP and IBMX (Figure 4). Moreover, W-7 blocked the transcription of lip and mnp isozymes in the presence of cAMP and IBMX (Figure 4) and did not affect intracellular cAMP concentration (Figure 5). All these data suggest that cAMP signaling increases LiP and MnP transcripts through the induction of cam transcription.




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