Effect of solid-medium coupled with reactive oxygen species on ganoderic acid biosynthesis and MAP kinase phosphorylation in Ganoderma lucidum Bang-Jau You a, ⁎, Wen-Te Chang a , Kuang-Ren Chung b , Yueh-Hsiung Kuo c , Chang-Syun Yang c , Ni Tien d , Hui-Chuan Hsieh a , Chun-Chien Lai a , Hong-Zin Lee e, ⁎ a Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung, Taiwan b Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, FL, USA c Tsuzuki Institute for Traditional Medicine, China Medical University, Taichung, Taiwan d Department of Laboratory Medicine, China Medical University Hospital, Taichung, Taiwan e School of Pharmacy, China Medical University, Taichung, Taiwan abstract article info Article history: Received 31 March 2012 Accepted 6 September 2012 Keywords: Mushroom Ganoderma lucidum Ganoderic acid biosynthesis Reactive oxygen species Signal transduction MAPK Ganoderma lucidum is used as a functional food and medicinal mushroom in Asia. Biosynthesis of its active ingredients, the ganoderic acids (GAs), together with signal transduction related to the fungal biology of G. lucidum, are critical for this application. However, regulation of G. lucidum GA biosynthesis and signal transduction remains enigmatic. We investigated the role of reactive oxygen species (ROS) on biosynthesis of individual GAs and on MAPK (mitogen-activated protein kinase) signaling. Antioxidant treatment de- creased lanosta-7,9(11), 24-trien-3α-01-26-oic acid (ganoderic acid 24) and total GA production. Cultures incubated with the pro-oxidant CDNB (1-chloro-2,4-dinitrobenzene) or H 2 O 2 , increased production of ganoderic acid 24 and total GAs were found. Interestingly, CDNB reduced RNA expression of the squalene synthase and lanosterol synthase, indicating that both genes may not be bottlenecks in GA induction. H 2 O 2 induced phosphorylation of the proteins Hog-1 and Fus3, which are homologs of the mammalian MAPKs p38 and ERK (extracellular signal-related kinases), respectively. CDNB also enhanced phosphorylation of Hog1 but had no effect on Fus3. To the best of our knowledge, this is the first report indicating that ROS reg- ulate the biosynthesis of individual GAs and affect MAPK signaling in G. lucidum. This study also identifies the potential application of ROS to the G. lucidum functional food industry. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction Ganoderma lucidum is a basidiomycete fungus that is considered to promote longevity and preserve vitality in Asia. In addition, G. lucidum has been used as a folk medicine in Asia for many centuries to treat human diseases such as chronic hepatitis, hypertension, cancer, in- flammation, and heart disease (Paterson, 2006). A number of func- tional food products of G. lucidum are popular in Taiwan in the form of drink, tea, and powder to enhance health, improve immunity, pro- tect liver and delay senility. Ganoderic acids (GAs) are one of the major ingredients which belong to triterpenoids and contribute to the pharmacological activity of G. lucidum. More than 130 triterpenoids have been reported and charac- terized in G. lucidum (Paterson, 2006; Shiao, 2003). Numerous biological activities of these triterpenoids have been identified including anticancer (Calviño et al., 2010; Yue et al., 2010), antiviral (El-Mekkawy et al., 1998; Min, Nakamura, Miyashiro, Bae, & Hattori, 1998), hepatoprotective (Kim, Shim, Kim, & Jang, 1999), antiplatelet aggregation (Wang, Chen, Shiao, & Wang, 1991), antioxidant (Heleno et al., 2012; Zhu, Chang, Wong, Chong, & Li, 1999), hypocholesterolemic (Hajjaj, Macé, Roberts, Niederberger, & Fay, 2005; Komoda, Shimizu, Sonoda, & Sato, 1989), and histamine release-inhibitory activity (Kohda et al., 1985). Biosynthesis of triterpenoids has been proposed via the mevalonate/ isoprenoid pathway starting by the conversion of acetyl CoA to synthesize mevalonate which subsequently becomes isopentenyl-pyrophosphate and then farnesyl diphosphate (Hirotani, Asaka, & Furuya, 1990; Shiao, 1992). Squalene synthase (SQS) and lanosterol synthase (LS) then cata- lyzes the formation of squalene and lanosterol, respectively (Shang, Shi, Ren, Qin, & Zhao, 2010; Zhao et al., 2007). It is then proposed that oxida- tion, reduction, hydroxylation, and dimethylation occur in order to syn- thesize specific GAs from lanosterol, but the details of these processes are still unknown (Liang et al., 2010). The fruiting body of G. lucidum is the major parts used for medical treatment. However, it is time-consuming to cultivate the fruiting body. As a result, submerged culture has been intensively studied as an alternative for producing fungal mycelium with high content of GAs (Tang, Zhang, Liu, Zhu, & Zhong, 2011; Tang, Zhang, & Zhong, 2009; Xu, Xu, & Zhong, 2010; Xu, Zhao, & Zhong, 2010). The optimiza- tion approaches that have been used to improve submerged culture Food Research International 49 (2012) 634–640 ⁎ Corresponding authors. Tel.: +886 4 22053366x5210; fax: +886 4 22078083. E-mail address: bangjau@mail.cmu.edu.tw (B.-J. You). 0963-9969/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2012.09.003 Contents lists available at SciVerse ScienceDirect Food Research International journal homepage: www.elsevier.com/locate/foodres