**1. Introduction**

*Lentinula edodes* (shiitake mushroom) is the second-most popular edible mushrooms in the world (the No. 1 is *Agaricus bisporus*), due to its high nutritional and medicinal values as well as the unique flavor [1–4]. Lenthionine (1,2,3,5,6-pentathiepane), the unique aroma of *L. edodes* [5,6], is derived from lentinic acid in a two-step enzymatic reaction [7,8]. In the reaction, the lentinic acid is catalyzed by γ-glutamyl transpeptidase (GGT) and L-cysteine sulfoxide lyase (C-S lyase) to generate the unique flavor compounds, including lenthionine [8–10]. Nevertheless, formaldehyde is also produced in this metabolic pathway (Figure 1).

Formaldehyde, a mutagen, can be found in the air, natural and processed foods, especially in frozen food and dry foods, and is classified as a human carcinogen by the International Agency for Research on Cancer (IARC) of World Health Organization [11]. The maximum daily dose reference for formaldehyde is defined as about 0.2 mg/kg body weight per day by the US Environmental Protection Agency. Even small doses of formaldehyde can cause various symptoms of physical discomfort [12]. However, the formaldehyde contents are 1–20 mg/kg in various fruits, vegetables, meat and fish

products [13]. In recent years, the amount of formaldehyde in *L. edodes* has raised the public concerns about food safety. For instance, high levels of formaldehyde (100–300 mg/kg) have been detected in shiitake mushroom samples produced in UK and Chinese [14]. Japanese researchers have reported that formaldehyde is generated in the growth process of *L. edodes* as a normal metabolite to form its unique flavor [15].

**Figure 1.** Proposed pathway for the generation of sulfurous flavor compounds and endogenous formaldehyde in *Lentinula edodes*.

Meanwhile, γ-glutamyl transpeptidase (GGT; EC 2.3.2.2) is an enzyme that catalyzes the transfer of the γ-glutamyl group of glutathione and related γ-glutamyl amides to water (hydrolysis) or to amino acids and peptides (transpeptidation) [16]. Cysteine sulfoxide lyase (EC 4.4.1.4) is a pyridoxal-5-phosphate (PLP) dependent enzyme and assigned to the class I family of PLP dependent enzymes [17]. In our previous work, the two enzymes were purified and characterized to play a significant biochemical role in the generation of endogenous formaldehyde in *L. edodes*[18]. Additionally, the gene of *Csl* encoding Lecsl (*L. edodes* C-S lyase) was cloned [19].

However, little is known about the relationship of GGT and C-S lyase with the production of formaldehyde in *L. edodes*. Thus, the aims of the present work were to determine the mRNA and protein expression levels and the activities of GGT and C-S lyase at different growth stages of shiitake mushrooms and to explore their correlations with endogenous formaldehyde production in *L. edodes*. This research could provide a molecular basis to understand the regulatory mechanisms of endogenous formaldehyde generation in *L. edodes* during the growth process.

#### **2. Results and Discussion**

#### *2.1. Gene Expression of Ggtl and Csl*

The expressions of *Ggtl* (encoding Leggt) and *Csl* (encoding Lecsl) during fruiting body development were analyzed by examining their transcript levels using real-time quantitative PCR. Both *Ggtl* and *Csl* were expressed at all the five stages of fruit-body development: mycelia, grey, young fruiting body, immature fruiting body and mature fruiting body, but differed in their expression patterns (Figure 2). Specifically, the transcript level of *Ggtl* decreased during the growth process, in contrast to an increase for *Csl*. Additionally, *Ggtl* showed the highest and lowest expression level in mycelia and mature fruiting body, respectively, which was just the opposite for *Csl*. There was approximately 1.5-fold difference between the highest and lowest expression levels in the two genes. Analysis of variance showed a significant difference in the *Ggtl* and *Csl* expression levels between mycelia and fruiting body stages, while *Ggtl* exhibited a significantly different expression in the four fruiting body stages (*p* < 0.05).

Based on our in-house transcriptome data, the expression pattern of *Ggtl* in different growth stages was basically in line with the quantitative RT-PCR results of our experiment. *Csl* was first reported as a gene involved in the generation of unique aroma of *L. edodes* [19]. In an early report, *Csl* displayed no obvious change in the expression level at 1, 2 and 3 h in the stage of mycelium or fruiting body during hot-air drying [20], which were basically consistent with our research results. Overall, there was a gradual decline of *Ggtl* expression level in different growth stages and an obvious increase of *Csl* expression level between mycelia and fruiting body stages.

**Figure 2.** Expression of *Ggtl* and *Csl* during fruit-body development of *Lentinula edodes* strain W1. Relative expression during five growth stages, M, mycelia; G, grey; YFB, young fruiting body; IFB, immature fruiting body; MFB, mature fruiting body. Transcript levels of *Ggtl* (black bars) and *Csl* (gray bars) were determined by real-time quantitative RT-PCR analysis and normalized against *Actinl*. The expressions during the M stage were taken as 1. Error bars indicate standard deviation for three independent experiments. \**p* < 0.05, ANOVA tests by Duncan's indicate significant differences.

#### *2.2. Western Blot of Leggt and Lecsl*

The protein expression levels of Leggt (GGT protein of *L. edodes*) and Lecsl (C-S lyase protein of *L. edodes*) in *L. edodes* at different growth stages were determined by Western blot using β-actin as an internal reference (Figure 3A,B) [21]. Notably, Leggt exhibited three bands, which conformed to previous reports [22,23], and their gray values showed a gradual decrease in the five stages. A previous study has shown that a mature gamma-glutamyl transpeptidase consists of one polypeptide chain and can be divided into a large and a small subunit by self-catalysis at the highly conserved threonine [24]. Correspondingly, three bands were shown in our Western blot analysis of Leggt. The protein expression levels showed a decrease in Leggt while a gradual increase in Lecsl during the growth process of *L. edodes* (Figure 3C). The overall trend of protein expression levels was similar to that of mRNA expression levels. Moreover, the two proteins showed significant differences in all the five samples. The changes between the highest and lowest expression levels of Leggt and Lecsl were 2.1-fold and 1.9-fold, respectively. This is the first report on the expression levels of these two enzyme proteins in shiitake mushrooms. Our data indicated that the expression of Leggt decreased while Lecsl increased across the five growth stages of *L. edodes*.
