**1. Introduction**

*Ganoderma lucidum* is a medicinal fungus that has been used to improve health and prevent certain diseases in Asia for thousands of years [1]. In modern ages, many bioactive compounds such as polysaccharides and triterpenoids [2,3] were identified and isolated from *G. lucidum*. These compounds were demonstrated to be effective for anti-cancer, anti-oxidant, anti-bacterial, anti-inflammation, and immune-regulation purposes [2,3].


Unlike triterpenoids from ginseng plants—which usually exist in the glycosidic form, called ginseng saponins—very few *Ganoderma* triterpenoid glycosides have been identified, despite the existence of many triterpenoids in *G. lucidum* [4]. The glycosidic form of triterpenoids might improve the bioactivity of the triterpenoids. For example, several ginseng saponins were found to exhibit more bioactivities involved in the central nervous system, cardiovascular system, and immune functions than ginseng triterpenoid aglycones were [5]. The glycosylation of flavonoids can also increase both water solubility and flavonoid stability [6–8]. It is, therefore, worthwhile to investigate the glycosylation of *Ganoderma* triterpenoids for potential medical and clinical purposes.

In nature, glycosylation is usually catalyzed by glycosyltransferase (GT, EC 2.4.x.y), a type of enzyme that uses a nucleotide-activated sugar donor, such as uridine diphosphate (UDP)-glucose, to transfer the sugar moiety to a sugar acceptor molecule [9]. Several GTs that catalyze the glycosylation of triterpenoids have already been discovered from plants, due to the accumulating knowledge on the metabolic pathways of triterpenoid glycosides [10]. However, plant GTs are not good candidates for the biotransformation of xenobiotics (such as *Ganoderma* triterpenoids) because plant GTs are usually involved in triterpenoid biosynthesis pathways and thus have very high substrate specificity. In contrast, GTs from bacterial sources usually have lower substrate specificity and have been demonstrated to be involved in the glycosylation of ginseng triterpenoids [11].

Among the *Ganoderma lucidum* bioactive compounds, ganoderic acid A (GAA) is one of the major triterpenoids and has been shown to prevent the proliferation of cancer cells and reduce inflammation activities [12–16]. Our previous study showed that the *Bacillus subtilis* ATCC (American type culture collection) 6633 strain can biotransform GAA into one major compound (**1**) and one minor compound (**2**) (Figure 1) [17]. In addition, two GTs—BsUGT398 and BsUGT489—were identified to catalyze the biotransformation of GAA into compound (**1**), which was later identified as GAA-15-*O*-β-glucoside [17]. However, the chemical structure of the compound (**2**) and its corresponding catalyzing enzyme remain elusive. In the present study, a GT enzyme that catalyzes the biotransformation of GAA to compound (**2**) was successfully identified, along with the optimal condition for producing compound (**2**) by the GT enzyme. The chemical structure of the previously-unknown compound (**2**) was also elucidated with the scaled-up production of the GT enzyme under an acidic condition.

**Figure 1.** Biotransformation of ganoderic acid A (GAA) by *Bacillus subtilis* ATCC 6633 after 24 h of incubation (solid line). The figure was modified from Figure 1 of our previous study [17].
