The biosynthesis of dihydrosphingosine (DhSph), involves two key enzyme-catalyzed reactions and is the starting point in the biosynthesis of all GSLs (Figure 2I). The first and rate-determining step is the condensation of palmitoyl coenzyme A (palmitoyl-CoA) and L-serine, catalyzed by serine palmitoyltransferase (SPT), to produce 3-keto dihydrosphingosine (3-keto DhSph), which is then reduced to DhSph by 3-keto DhSph reductase (KDSR) (Barreto-Bergter et al., 2004). The genes encoding SPT and KDSR are conserved among all organisms and have been identified from various species of fungi. Two genes, LCB1 and LCB2, which are necessary for SPT activity, have been identified in Saccharomyces cerevisiae (Buede et al., 1991; Nagiec et al., 1994). Deletion of either LCB1 or LCB2 was found to abolish GSL production in yeast (Mukherjee and Dekker, 1990; Zhao et al., 1994; Dickson, 2008). In A. nidulans, the gene encoding SPT was identified and named lcbA owing to its homology to the S. cerevisiae LCB1. The lcbA is essential for polarity and growth in A. nidulans and under the control of the alcA promoter, which is alcohol inducible and glucose repressible (Waring et al., 1989; Cheng et al., 2001). An important third subunit of SPT is Tsc3p, which coimmunoprecipitates with LCB1 or/and LCB2, and is required for optimal and high level SPT activity during maximal GSL biosynthesis in S. cerevisiae (Gable et al., 2000).

The role of 3-keto DhSph reductase remains poorly investigated in filamentous fungi. In A. fumigatus, KDSR is encoded by the ksrA gene, but the biological role of ksrA in Aspergillus remains to be elucidated (Fornarotto et al., 2006). Deletion of C. albicans ksr1 shows compromised filamentation, suggesting that the expression of ksr1 may be important for polarized growth (Fornarotto et al., 2006).

Although poorly studied, the production of DhSph is a critical and common pathway in the biosynthesis of neutral and acidic GSLs. This compound can generate two distinct ceramide pools of dihydroceramide and phytoceramide, which are utilized for the formation of neutral and acidic GSLs, respectively. Two genes, BarA and BasA, are involved in this step in filamentous fungi (Li et al., 2006; Rittenour et al., 2011; Cheon et al., 2012; Fontaine, 2017). The ceramide pool involved in the neutral GSL synthesis (dihydroceramide) is generated by BarA, while acidic GSL synthesis (phytoceramide) is catalyzed by BasA (also called Sur2 in S. cerevisiae). Therefore, filamentous fungi possess two distinct ceramide pools that make independent contributions to polarized hyphal growth, through the formation of specialized lipid microdomains that regulate the organization of the cytoskeleton (Li et al., 2006).

Synthesis of the neutral GlcCer and GalCer from DhSph includes four enzyme-catalyzed reactions (Figure 2II).

The synthesis of acidic GSLs, including the productions of IPCs, MIPCs, and M(IP)₂Cs, also starts from DhSph and includes three enzyme-catalyzed reactions (Figure 2III).

Blocking GSL biosynthesis has become a target for developing antifungal therapies and understanding fungal biology. Thus, enzyme inhibitors of synthetic GSLs for a variety of biological processes have already been demonstrated. There are five key steps in which enzymes can be blocked by inhibitors in the GSL biosynthetic pathway (Figure 2 the blue fonts). As the first rate-limiting enzyme of the GSL biosynthetic pathway, SPT has been the subject of many studies to identify inhibitors. One of the earliest SPT inhibitors identified is the mechanism-based inhibitor L-cysteine (Zheng et al., 1994, 1998). The most widely used and studied natural product SPT inhibitors are myriocin and sphingofungin that have been shown to block biofilm formation and polarized growth of fungal hyphae in Candida and Aspergillus species (Cheng et al., 2001; Lattif et al., 2011; Perdoni et al., 2015; Harrison et al., 2018). In addition, β-chloroalanine, β-fluoroalanine, and halide can also be used to inhibit SPT (Lev and Milford, 1981; Medlock and Merrill, 1988; Smith and Merrill, 1995). Fumonisin B1 and australifungin are inhibitors of ceramide synthase that alter GSL metabolism by inhibiting ceramide synthesis, and act as antifungal agents in Cryptococcus, Candida, and Aspergillus species (Mandala et al., 1995; Merrill et al., 2001). Glucosylceramide synthase is involved in the final step of GlcCer synthesis and can be blocked by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threoPDMP) that cause a reduction in hyphal germination and colony growth in A. nidulans, A. fumigatus, and Aspergillus terreus (Levery et al., 2002).

Inositol phosphorylceramides synthases are unique structures in fungi involved in many cellular processes, including growth, differentiation, and morphogenesis, but are not found in mammals (Barr and Lester, 1984; Levery et al., 1998, 2002; Loureiro y Penha et al., 2001). Therefore, IPC synthases constitute potential targets for the development of new antifungal drugs, as inhibiting it will lead to the accumulation of PhSph and PhCer and, ultimately fungal death. Aureobasidin A, khakrefungin, and galbonolide inhibitors of IPC synthase can block transfer of myo-inositol-1-phosphate from PI to ceramide, resulting in accumulation of PhSph and PhCer, and complete inhibition of IPC production, along with cell cycle arrest in C. albicans, Cryptococcus neoformans, S. cerevisiae, and A. nidulans (Cheng et al., 2001; Cerantola et al., 2009; Tan and Tay, 2013). Therefore, IPC inhibitors, with low toxicity in mammals due to the lack of a mammalian IPC synthase, might be possible candidates as ideal antifungal drugs (Takesako et al., 1993; Mandala et al., 1997, 1998; Georgopapadakou, 2000; Rollin-Pinheiro et al., 2016).

Glycosphingolipids are the main lipid components of microdomains and are clustered along with sterols in specialized membrane microdomains to form lipid rafts in filamentous fungi (Sonnino et al., 2007). These regions play crucial roles in cell polarization and hyphal growth, mediated by the cytoskeleton and accumulation of lipid raft components at the growing tip of hyphal cells (Cheng et al., 2001; Barreto-Bergter et al., 2004; Pearson et al., 2004; Nimrichter and Rodrigues, 2011; Guimarães et al., 2014). Biological functions of GSLs have been studied through gene deletion and inhibitory approaches in filamentous fungi (Rittershaus et al., 2006; Oura and Kajiwara, 2008, 2010; Ramamoorthy et al., 2009; Zhu et al., 2014; Fernandes et al., 2016). Several studies have demonstrated that impairment of any steps of GlcCer biosynthetic pathway leads to compromised mycelium growth, hyphal elongation, and lipid raft mislocalization. For example, disruption of the initial step of GlcCer synthesis by deletion of barA or by inhibition in presence of HSAF strongly reduces polarized growth and leads to the formation of enlarged and hyperbranched hyphae, with altered cell wall (Li et al., 2006, Li et al., 2009). Similarly, disruption of the final step of GlcCer synthesis by deletion of GCS or by glucosylceramide synthase inhibitors also impairs hyphal extension in A. nidulans and inhibits morphological transitions such as budding and germ tube formation in A. fumigatus (Levery et al., 2002; Ramamoorthy et al., 2007; Fernandes et al., 2016). These results suggest that GlcCer synthase is essential for the establishment and maintenance of the polarity axis, fungal growth, and differentiation in filamentous fungi (Figure 3).

In filamentous fungi, not only is growth and differentiation reduced upon impairment of neutral GSL synthesis, but growth also relies on the production of IPC (Figure 3). Inhibition of IPC synthase is followed by an accumulation of PhSph and PhCer intermediates that lead to fungal cell cycle arrest and apoptosis (Cheng et al., 2001). Integrity of the IPC synthesis pathway is relevant for filamentous fungal viability, not only due to the role of IPC in fungal differentiation but also due to the need for highly regulation of DhSph, PhSph, and PhCer levels, as DhSph and PhSph are highly toxic to fungal cells. Thus, changes in their levels may have uncontrolled effects on signaling events, resulting in fungal cell death which indicate that PhCer synthase and IPC synthase can be potential targets for development of new antifungal drugs, as inhibiting these enzymes will lead to the accumulation of PhSph and PhCer, and, ultimately, fungal death.

In addition to fungal polarization and hyphal growth, GSLs also play important role in filamentous fungal fitness and adaptation to diverse environments as a signaling molecules (Grimm et al., 2006; Lingwood and Simons, 2010; Figure 3). The fungal heat shock response is characterized by the production of heat shock proteins that function as chaperonins, and in the processes of protein degradation. GSLs possess the capacity to serve as bioactive signaling molecules in early heat stress response. This signaling function influences the regulation of the cell cycle and the synthesis of heat shock proteins, which have important secondary effects, especially if heat shock proteins are not available to serve as protectors of other proteins (Jenkins, 2003). For example, C. albicans treatment with inhibitor of SPT, leads to the decrease in lipid domain accumulation, resulting in less recruitment of heat-shock proteins (Insenser et al., 2006). In addition, environmental stresses directly affect the representation of fungal GSL classes (Řezanka et al., 2018). Thermophilic microorganisms can change the contents of IPC, MIPC, or M(IP)₂C to mediate fungal apoptosis in response to high temperature stress (Řezanka et al., 2016, 2018). N. crassa synthesizes PhCer as signal molecules to mediate fungal apoptosis in response to heat stress (Plesofsky et al., 2008). In S. cerevisiae, the accumulation of IPC is detrimental to yeast under low pH conditions, and downregulation of IPC levels is one of the adaptation mechanisms for low pH conditions (Otsu et al., 2020). In contrast, reduction of the IPC level increases the sensitivity of C. neoformans to low pH and impairs the growth and the pathogenicity of C. neoformans (Luberto, 2001). Besides, blocking the pathway of GSL synthesis or adding exogenous GSLs can significantly affect fungal tolerance to acidic or alkaline environmental stress. For example, in C. neoformans, the Δcer1 mutant cannot survive in acid or alkaline environments due to its inability to synthesize GlcCer (Ishibashi et al., 2012; Munshi et al., 2018). Besides, the C. neoformansΔsld8 mutant that only synthesizes saturated GlcCer is more susceptible to membrane stressors and shows increased membrane permeability, resulting in decreased stress resistance (Raj et al., 2017). Similarly, S. cerevisiae, which is incapable of synthesizing GlcCer, can adapt to alkaline and ethanol stress after exposure to Aspergillus kawachii-derived GlcCer via altering the yeast membrane properties by exogenous GlcCer (Sawada et al., 2015). These results demonstrate that GSLs play important roles in response to environmental stress in fungi (Figure 3).

Impairment of polarized hyphal growth and mislocalization of lipid microdomains were observed when the biosynthetic steps of sphingolipid desaturase and sphingolipid C₉-methyltransferase were interrupted, demonstrating that unsaturation and C₉-methylation of the neutral GSLs are essential for growth and differentiation in filamentous fungi. The structures of Δ4,Δ8-unsaturated and C₉-methylated LCB, distinguishing filamentous fungi from plant and mammalian sphingosines, are unique to fungal neutral GSLs (Barreto-Bergter et al., 2004, 2011; Del Poeta et al., 2014). Disruption of the A. nidulans sdeA leads to increase accumulation of saturated and unmethylated GlcCer and reduce mycelium growth (Fernandes et al., 2016). Previous studies in C. neoformans showed that Δsld8 mutant synthesizing only saturated GlcCer is more susceptible to membrane stressors follow by increasing membrane permeability, even though saturated GlcCer produced more lipid rafts than unsaturated GlcCer species (Raj et al., 2017). The C₉-methylated found in LCB is required for normal growth and differentiation in filamentous fungi (Ramamoorthy et al., 2009; Fernandes et al., 2016). A. nidulans smtA deletion combined with conditional repression of smtB significantly increases unmethylated GlcCer levels and compromises filamentous growth (Fernandes et al., 2016). The ΔFgmt2 mutant produces 65–75% unmethylated GlcCer shows severe growth defects when compared to the wild-type strain (Ramamoorthy et al., 2009). In pathogenic yeasts, deletion of the gene encoding C₉-methyltransferase results in a mutant with attenuated virulence (Noble et al., 2010; Singh et al., 2012). Interestingly, certain plant defensins require the C₉-methylation for fungal GSL recognition (Oguro et al., 2014; Fernandes et al., 2016), which suggest that plant defensins may have a therapeutic potential for treatment of fungal infections for fungus specific C₉-methylation (Figure 3).

The unsaturated FAs of neutral GSLs are also important for the mycelial growth in filamentous fungi. The Δ3-unsaturated FA is a unique modification of fungal GSLs, which has been reported in neutral GSLs of A. oryzae (Yasuhiko and Masao, 1997), A. fumigatus (Toledo et al., 1999), and S. schenckii (Toledo et al., 2001), and is involved in signaling pathways that control morphological switch (Figure 3). In fact, the ratio of saturated and Δ3-unsaturated 2-hydroxy FAs vary among the GSLs from different fungal morphotypes. For example, only 15% of GlcCer extracted from the Paracoccidioides brasiliensis yeasts is composed of Δ3-unsaturated FAs, while 50% of GlcCer contains the Δ3-unsaturation in P. brasiliensis mycelium (Toledo et al., 1999). Similarly, GlcCer from Histoplasma capsulatum yeast contains a higher proportion of saturated FAs, while the GlcCer from mycelium is almost exclusively constituted by the Δ3-unsaturated FAs (Toledo et al., 2001). The higher contents of Δ3-unsaturated GlcCer in mycelial forms of P. brasiliensis and H. capsulatum may be ascribed to the activation of desaturase activity during the yeast-to-hypha transition, suggesting that Δ3-unsaturation of GlcCer may be involved in signaling pathways that control morphological switch.

In contrast to GlcCer, most studies of GalCer have been performed in mammals and its biological function in filamentous fungi remains unknown. It has been previously shown that sphingolipids regulate the activity of protein kinases, such as protein kinase C and protein phosphatases, involved in signaling cascades that ultimately modulate cell growth, differentiation, and proliferation (Hannun and Obeid, 2008). S. schenckii mycelia synthesize only GlcCer, while yeast forms are found to contain both GlcCer and GalCer, suggesting that the ceramide galactosyltransferase may be activated during the S. schenckii mycelium-yeast switch or inhibited during the yeast-to-hypha transition (Dickson and Lester, 1999; Toledo et al., 2000). Structural analyses of neutral GSLs between A. fumigatus and S. schenckii show that GalCer and GlcCer possess identical ceramide backbones, except that GalCer contains a higher proportion of Δ3-unsaturated FA (Toledo et al., 1999, 2000). Similarly, the GalCer production and differential Δ3-unsaturation of FAs may constitute a molecular mechanism of GSL control over fungal morphogenesis through the activation/inactivation of signal transduction pathways (Fernandes et al., 2018).

Ceramide is thought to be a critical molecule in the maintenance and formation of the epidermal permeability barrier and an important component in water-retention of skin (Hamanaka et al., 2005). GlcCer can be hydrolyzed to ceramide by β-glucocerebrosidase in keratinocytes, and the resultant ceramide is stored in stratum corneum (Figure 4). Levels of GlcCer significantly increase during epidermal differentiation, and then GlcCer is enzymatically hydrolyzed to ceramides to regulate permeability barrier function. The level of epidermal ceramide is regulated by a balance between β-glucocerebrosidase, sphingomyelinase, and ceramidase (Holleran et al., 2006). A deficiency or inhibition of β-glucocerebrosidase in the epidermis can alter the distribution of ceramide and GlcCer, resulting in decreased the epidermal permeability barrier function (Alessandrini et al., 2004; Carneiro et al., 2011). Low levels of GluCer and ceramide in atherosclerosis-prone mice led to skin inflammation and hair discoloration and loss (Bedja et al., 2018). Prior studies demonstrated that all molecular species of stratum corneum ceramides are derived from GlcCer (Uchida et al., 2000; Hamanaka et al., 2002). In addition, GlcCer also prevents dehydration of the stratum corneum and repaired the barrier function of skin. Therefore, the GlcCers play critical role in enhancing the epidermal permeability barrier function.

Furthermore, the beneficial effects of oral intake of plant GlcCer for skin hydration and skin barrier reinforcement have also been established in several studies involving animal models (Tsuji et al., 2006; Uchiyama et al., 2008; Yeom et al., 2012; Kawano and Umemura, 2013) as well as human subjects (Miyanishi et al., 2005; Kimata, 2006; Uchiyama et al., 2008; Guillou et al., 2011; Figure 4). The present studies suggest that the lipophilic fraction of GlcCer, present in plants has protective effects against intestinal impairment, but it requires extraction since digestion alone is not enough to elicit its complete protective action. Therefore, purified GlcCers are usually added in health food as a function complement. Some studies report that an intake of 0.6–1.8 mg/day of GlcCer from supplements is enough to improve human skin health (Uchiyama et al., 2008; Hirakawa et al., 2013). Shimoda et al. (2012) established the effects of GlcCer on the changes in epidermal ceramide and GlcCer in mice after oral intake of rice-derived GlcCer, as well as in a human epidermal equivalent. These findings demonstrated an increase in the level of epidermal ceramide follow by a decrease in the amounts of GlcCer (accompanied with enhanced β-glucocerebrosidase and GlcCer synthase expressions), suggesting epidermal GlcCer metabolism enhancing effect by oral intake of GlcCers. Oral administration of GluCer from maize significantly reduced UVA-induced wrinkle formation in the skin as well as epidermal hypertrophy of hairless mice (Shimada et al., 2011). Oral intake of konjac GlcCer has been reported to decrease the transepidermal water loss (TEWL) in atopic dermatitis (AD) patients (Miyanishi et al., 2005), and improve skin symptoms (including TEWL reduction) and reduce skin allergic responses in children with moderate AD (Kimata, 2006). These studies support the beneficial effects of oral intake of plant GlcCer and their potential complementary and alternative therapeutic applications in the restoration and maintenance of skin barrier function. Therefore, GlcCers exhibit a wide potential for development and application as an important biological resource in improving epidermal barrier function.

In addition to prevent AD and improved skin-barrier function, plant GlcCer also prevent intestinal impairment as dietary supplements (Figure 4). Recently, the incidence rate of intestinal impairments, such as colon cancer and inflammatory bowel disease (IBD), has increased in East Asian countries and Western countries (Molodecky et al., 2012). It is difficult to recover completely from IBD and these patients have an increased risk of developing colon cancer (Triantafillidis et al., 2009). There are many studies on the effect of GlcCer contained in food on intestinal impairment. In vitro experiments indicate the possibility that GlcCer protects the colon surface from the harmful effects of various drugs (Yamashita et al., 2017). In addition, GlcCer has been shown to have an apoptosis-inducing effect on colon cancer cells in vitro (Aida et al., 2004). Previous studies demonstrate that uptake of dietary GlcCer may improve the microenvironment of intestinal tract via modulating the intestinal microbiota (Schmelz et al., 2015). Yamashita et al. (2019) had confirmed that dietary GlcCer can significantly suppress aberrant crypt foci (ACF) formation and the production of inflammation-related cytokines in mice fed with rice-derived extracted GlcCer, which suggest GlcCer extracted from polished rice has protective effects against intestinal impairment. Collectively, dietary GlcCers can be digested by the intestinal microbial flora and display preventive and chemotherapeutic effects on colon cancer in animal models; however, clinical trials are urgently needed to investigate the response of colon carcinogenesis to dietary GlcCer in the future.

Although there are few studies on the applications of fungal glycosylceramides, we summarized several applications of them in cosmetics or foods. Interestingly, Takahashi confirmed that GlcCer produced by A. oryzae constituted the most abundant species (43% of the total GlcCer) in the sake lees, which is brewed with A. oryzae and S. cerevisiae, and has long been recognized for its moisture-holding activity in cosmetics in Japan (Takahashi et al., 2014). Torula yeast (Candida utilis)-derived GlcCer has been reported to increase dermal fibroblast proliferation and collagen production to maintain dermal elasticity (Fukunaga et al., 2019). Miyagawa et al. (2019) investigated the effects of glycosylceramides on gene expression in normal human epidermal keratinocytes, which reveal that koji and Aspergillus luchuensis and A. oryzae glycosylceramides increased the expression of occludin (OCLN, an epidermal tight junction protein) and ATP-binding cassette sub-family A member 12 (ABCA12, a cellular membrane transporter) to increase ceramide in the keratinocytes. These results indicated that glycosylceramides have an effect of increasing genes expression which involved in skin barrier function and the transport of lipids in the keratinocytes, and suggest that koji exerts its cosmetic effect by increasing ceramide and tight junctions via glycosylceramides. In addition to function in cosmetics, fungal neutral GSLs also play an important role in foods. For example, Ferdouse et al. (2018) have proved that addition of A. oryzae glycosylceramide can affect the flavor and metabolic profiles of sake yeast in the manufacture of sake. They also demonstrate that addition of A. oryzae, Glycine max, and Grifola frondosa GlcCers confer a similar effect on the flavor profiles of sake yeast (Ferdouse et al., 2018), which indicate that the effects of plant GlcCer and A. oryzae GlcCer on flavor profiles are similar. In addition, A. oryzae glycosylceramide, used as a prebiotic in obese mice, can be digested by the intestinal microbial flora and increased ratio of B. coccoides, which is a potentially beneficial species in the intestine (Hamajima et al., 2019). Besides, feeding of A. oryzae glycosylceramide to obese mice can alter the cholesterol metabolism that liver cholesterol is significantly decreased in obese mice fed with A. oryzae glycosylceramide (Figure 4). These results will be of value in the utilization of fungal GlcCer and GalCer for cosmetics and functional foods.

Although neutral GSLs are originally derived from soybean and bovine sources, currently almost all the GlcCers used in cosmetics or health foods are extracted from plants. GlcCers from wheat flour (Ohnishi et al., 1985), potatoes (Bartke et al., 2006), maize, rice (Sugawara et al., 2010), and konjac (Uchiyama et al., 2008; Usuki et al., 2015) are now widely added in cosmetics as moisturizing agents or in health foods as “functional components.” The most abundant classes of GSLs in plant tissue are mono-GlcCers. In contrast, the GalCer of glycosylceramides is rarely detected or reported in plants (Sullards et al., 2000; Spassieva and Hille, 2003). In cosmetics, GlcCer must be hydrolyzed to ceramide that is then deposited in stratum corneum to form the epidermal barrier. The more selective and efficient approach of transforming GlcCer to ceramide is enzymatic hydrolysis of β-glucocerebrosidase. Interestingly, GalCer, restricted to mainly in neural tissues in mammals, can activate β-glucocerebrosidase to hydrolyze GlcCer in keratinocytes and increase ceramide content to improve dry skin and AD (Alessandrini et al., 2004). However, GalCer is mainly extracted from neural tissues of animals such as cows and has been for the subject of several studies until the discovery of bovine spongiform encephalopathy. Although the animal glycosylceramides obtained from bovine brain and biotechnological sources have been investigated, the safety profile for cosmetic and food applications has not yet been established (Adam, 2001; Ono et al., 2010). Thus, GalCer from neural tissues of animals is not acceptable for cosmetic or other human use due to the underlying risk for prion diseases. As an alternative, glycosylceramides isolated from edible plants are highly safe and preferable for cosmetic and therapeutic applications. Unfortunately, plant glycosylceramides only contain GlcCer and are totally devoid of GalCer. Therefore, it is important to seek new biological resources that can synthesize both GlcCer and GalCer, along with attempts to improve their contents by modifying the biosynthetic pathway of neutral GSLs using genetic engineering technology in known resources.

Glycosylceramides, which have now been commercialized as moisturizing agents or dietary supplements for dry skin, are mainly sourced from edible plants that only contain GlcCer and are completely without GalCer. Surprisingly, there are many reports that filamentous fungi Aspergillus species, such as A. oryzae, A. awamori, and A. sojae that are used in various Japanese fermented foods and drinks, can produce both GlcCer and GalCer (Fujino and Ohnishi, 1977; Zhang et al., 2007; Hirata et al., 2012; Tani et al., 2014). Therefore, glycosylceramides from Aspergillus that include both GlcCer and GalCer can make up for the lack of GalCer in plants and would be more effective for their barrier function of skin in cosmetics as compared to plant GlcCer, due to the GalCer can activate β-glucocerebrosidase to promote the hydrolysis of GlcCer to ceramide (Figure 4; Alessandrini et al., 2004; Tani et al., 2014). Furthermore, previous studies have elucidated that koji (A. oryzae, A. awamori, and A. sojae) contains abundant glycosylceramides (0.5–2 mg/g dry weight koji), which are comprised of GalCer (30.3%) and GlcCer (69.7%), and are one of the highest amounts found in any cuisine (Hirata et al., 2012; Hamajima et al., 2016). Therefore, since filamentous fungi Aspergillus are economically important in the industrial production and can synthesize both GlcCer and GalCer, they would be new sources for exploiting neutral glycosylceramides in future.

Another advantage of filamentous fungi in the production of GlcCer and GalCer is that the cell growth cycle of fungi is much shorter than that of plants, which can provide a large amount of mycelium for extraction of GlcCer and GalCer. In addition, most filamentous fungi used in the production of fermented foods are safe for humans, especially A. oryzae, which is generally regarded as safe according to the FDA and has been used for making fermented foods such as saké, shoyu (soy sauce), and miso (soybean paste) for thousands of years. Therefore, the A. oryzae would be a suitable source for production of GlcCer and GalCer due to their food safety. Furthermore, the availability of genome sequences of A. oryzae and the availability of DNA arrays, GeneChips, and RNA sequence have provided an unprecedented resource for studying and modifying the biosynthetic pathways of GlcCer and GalCer. In summary, we believe that filamentous fungi would be important resources for exploiting neutral glycosylceramides in the future, especially GalCer.