Concurrently Produce a Novel High-Potency Sweetener (Glycyrrhetic Acid 3-O-Mono-β-D-glucuronide) and Lignocellulolytic Enzymes using Plant Entophytic Chaetomium Globosum DX-THS3 by Solid-State Fermentation

Licorice straw was used for the rst time as a medium for glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG) and lignocellulosic enzyme production via solid-state fermentation (SSF) of endophytic fungus Chaetomium globosum DX-THS3. Under optimal fermentation conditions, the percent conversion of glycyrrhizin reached 90% in 15 days, whereas the control needed 35 days to achieve the same result. The productivity of optimization (P=2.1 mg•g -1 •day -1 ) was 2.33-fold that of non-optimization (P=0.9 mg•g 1 •day -1 ). Meanwhile, high activities of lter paper enzyme (FPase) (234.6 U/g), carboxymethyl cellulase (CMCase) (29.25 U/g), xylanase (72.52 U/g), and β-glucuronidase activity (264.17 U/g) were obtained faster than those in the control during SSF. Our study provides a novel and ecient strategy for GAMG production and indicates C. globosum DX-THS3 as a potential producer of lignocellulolytic enzymes.


Introduction
Glycyrrhizin (GL) is a major bioactive component of industrial crop Glycyrrhiza (Pandey & Ayangla, 2017) with numerous valuable physiological properties, including antioxidative (Michaelis et al., 2011), antiviral (Ito et al., 1988), anticancer (Huang et al., 2016), and anti-in ammatory (Wang et al., 2015a) action. GL can e ciently inhibit the coronavirus disease (COVID- 19) virus, and it is commonly considered a potential chemical for the cure of COVID-19 (Bailly & Gérard Vergoten, 2020;Murck, 2020). However, GL has low absorption and strong adverse effects in humans and animals (Akao, 2000; Wang et al., 2013). GL can be transformed into glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG), which possesses improved biological activities and safety and higher solubility, by hydrolysis of one of the terminal glucuronic acids of GL (Lin et al., 2009;Li et al., 2017). Furthermore, GAMG is about 5 and 1000 times sweeter than GL and sucrose (Mizutani et al., 1994), respectively, and tastes better (less aftertaste) than GL. Therefore, GAMG has valuable application, especially in the food industry. However, e cient approaches for large-scale GAMG production are unavailable thus far. GAMG can be produced by chemical synthesis and biotransformation. However, chemical approaches generally present several disadvantages, including the requirement of strong and harsh conditions, ineffective costs, poor selectivity, and environmental pollution (Brieskorn & Lang, 1978). Compared with chemical approaches, the biosynthesis of GAMG signi cantly has more potential and advantages because of its excellent substrate selectivity, high yields, mild reaction conditions, and eco-friendly status. has several challenges, such as complicated processes and harsh cultivation conditions. Therefore, an e cient, simple, and feasible approach for the production of GAMG must be developed.
In general, most enzymes are produced by microorganic submerged fermentation (SF). GUS can also be produced by SF of Aspergillus terreus Li-20 , Streptococcus LJ-22 (Park et al., 2005), and Penicillium purpurogenum Li-3 (Zou et al., 2013). However, their applications in the manufacturing industry have been met with several processing challenges, including low productivity, especially in terms of ineffective costs of the mass production of enzymes. Compared with SF, solid-state fermentation (SSF) can offer several advantages, including low equipment requirements, simple culture processing, and the use of widely available and inexpensive lignocellulosic residues (usually crop straws and wastes) as substrates (Pandey, 2003). Therefore, SSF is very suitable to produce highly bioactive products, such as enzymes. Thus, the utilization of SSF for large-scale production of GAMG should be considered.
However, to date, the use of SSF to produce GAMG has not been investigated.
Endophytic fungi are an ecological group of fungi that is present in living plant tissues without initiating any external symptoms. Endophytic fungi have attracted research attention because they can secrete a number of secondary metabolites, including bioactive chemicals (Suryanarayanan et al., 2012). In addition, endophytic fungi can be utilized as plant material, for example, crop straw, for use as a medium for SSF to produce valuable products, such as bioactive chemicals and enzymes (Prajapati et al., 2018;Natália et al., 2018;Deswal et al., 2011). In our previous study, an endophytic fungus Chaetomium globosum DX-THS3 was isolated from Dongxiang wild rice (Oryza ru pogon Griff.) (Wang et al., 2015b). Furthermore, a GUS with speci city and highly transformable GL to generate GAMG was screened from C. globosum DX-THS3 (Zhang et al., 2020), and genome analysis showed abundant genes coding lignocellulose-degrading enzymes harbored in the C. globosum DX-THS3 genome (unpublished). Therefore, in this study, C. globosum DX-THS3 was utilized with licorice straw as a medium for SSF. We aimed to i.) investigate the feasibility of GAMG production by C. globosum DX-THS3 via SSF and provide an e cient strategy for large-scale production of GAMG; ii.) analyze the lignocellulose-degrading enzymes, such as carboxymethyl cellulase (CMCase), FPase, xylanase, and GUS, during SSF to simultaneously obtain high-activity enzymes and demonstrate C. globosum DX-THS3 as a potential candidate for producing lignocellulose-degrading enzymes.

Chemicals and strains
Licorice straw was purchased from Inner Mongolia, China. Standard GL and GA samples (purity ≥ 98%) were purchased from Sigma Chemical Co. (USA). Standard GAMG (purity > 98%) was prepared and identi ed via carbon-13 nuclear magnetic resonance in our laboratory. Other chemicals and solvents (analytical grade) were purchased from Xilong Scienti c Co., Ltd. (China). The endophytic fungal strain C. globosum DX-THS3 (CCTCC M2016005) was isolated from healthy Dongxiang wild rice in a nature reserve in Dongxiang county, Jiangxi province, China in our previous work and collected by Jiangxi Normal University, Nanchang, China.

Production of GAMG by SSF
Licorice straw was dried for 24 h in a drying oven and then crushed to obtain < 3 mm grain diameter. C. globosum DX-THS3 was cultured in a 500 mL ask with 200 mL seed culture medium (potato dextrose broth (PDB)) at 28 °C on a rotary shaker at 150 rpm for seed broth preparation. Then, 2 mL seed broth was cultured in 15 g sterile licorice straw grain, which was pre-added with 5 mL sterile water at 28 °C for the production of GAMG. After 20 days of culture, 2 g culture medium was ground and added to 100 mL water for the determination of GAMG using thin layer chromatography (TLC) and ultraperformance liquid chromatography (UPLC, Waters, USA).

Optimization of SSF conditions
Strain DX-THS3 was cultivated in different fermentation conditions to detect the GUS activity and yield of GAMG for optimization of fermentation conditions. First, 2 mL seed broth was cultivated in licorice straw of different particle sizes (grain diameter: 0-0.25, 0.26-0.85, and 0.86-3.0 mm) at 28 °C. Then, the GUS activity and yield of GAMG were analyzed after culturing for 20 days. Then, strain DX-THS3 was grown on licorice straw at 24 °C, 28 °C, and 33 ℃ for the analysis of GUS activity and yield of GAMG after 20 days. For optimization of seed age, different seed broths cultivated in PDB medium for 48, 60, 72, 84, 96, 108, 120, and 132 h were grown on licorice straw at 28 °C to analyze the GUS activity and yield of GAMA after culturing for 20 days. To optimize the inoculum of seed broth, different volumes of seed broths (15%, 20%, 25%, 30%, 35%, and 40%, v/w) in licorice straw at 28 °C to analyze the GUS activity and yield of GAMA after culturing for 20 days. To optimize the water content of licorice straw, strain DX-THS3 were cultured on licorice straw containing different water contents (straw: water = 1:2, 1:2.5, 1:3, 1:3.5, m/m) at 28 °C to analyze the GUS activity and yield of GAMA after 20 days of culture. Finally, glucose, fructose, sucrose, lactose, NH 4 NO 3 , peptone, yeast powder, and yeast extract were added to the licorice straw for optimization of carbon and nitrogen sources. All cultivations were performed in triplicate.

Determination of lignocellulosic enzyme activities
Two grams of solid-state medium was sampled, ground by liquid nitrogen, and then dissolved in 15 mL sodium acetate (pH 5.0). The suspension was centrifuged at 4 °C and 10,000 × g, and the supernatant was collected for the preparation of crude enzymes. Endoglucanase activity (CMCase) was assayed in a reaction containing 1 mL carboxymethyl cellulose solution (2% sodium acetate, pH5.0, v/v) and 1 mL crude enzymatic solution. After incubation for 30 min at 50 °C, reducing sugar was detected by 3,5dinitrosalicylic acid (DNS) method (Zhao et al., 2008) with glucose as the standard. FPase activity was analyzed using Whatman No. 1 lter paper (1 × 6 cm 2 , 50 mg) in a 2 mL total volume reaction containing 1 mL crude enzymatic solution and 1 mL sodium acetate (pH 5.0) for 60 min at 50 °C. Then, the reducing sugar was detected by DNS method. One unit (U) of CMCase and FPase activity was de ned as the amount of enzyme that released 1 µmol glucose equivalent per minute. Xylanase activity was assayed in a solution containing 1% (w/v) xylan (1 mL) in 50 mM solidum acetate buffer (pH 5.0) and appropriately diluted crude enzyme (1 mL). After 10 min incubation at 50 °C, reducing sugar was detected by DNS method. One unit (U) of xylanase activity was de ned as the amount of enzyme that released 1 µmol xylose equivalents per minute. β-Glucosidase activity was detected based on the release of ρ-nitrophenol (ρNP) from the ρ-nitrophenyl-β-d-glucopyranoside (ρNPG) substrate, and the absorbance was read at 430 nm. The assayed reaction containing 1 mL crude enzyme and 1 mL ρNPG (5 mM) was incubated for 10 min at 50 °C and then disrupted by 3 mL 0.5 M sodium carbonate. One unit (U) of β-glucosidase activity was de ned as the amount of enzyme that released 1 µmol ρNP equivalent per minute.

Determination of total and reducing sugars
Two grams of solid-state medium was sampled, ground by liquid nitrogen, and then dissolved in 15 mL water. The suspension was centrifuged at 4 °C and 10,000 × g, and the supernatant was collected for sample preparation. The total sugar of solid-state medium was determined in accordance with the phenol sulfuric acid method (Masuko et al., 2005), whereas reducing sugars were quanti ed by the DNS method.

Determination of GUS activity
Two grams of solid-state medium was sampled, ground by liquid nitrogen, and then dissolved in 15 mL sodium acetate (pH 6.0). The suspension was centrifuged at 4 °C and 10,000 × g, and the supernatant was collected for the preparation of crude enzymes. GUS activity was assayed by the reaction containing 200 µL crude enzymatic solution and 800 µL GL solution (2 g/L). Then, the reaction was incubated at 45°C for 1 h and disrupted by boiling water and analyzed by UPLC. One unit (U) of GUS activity was de ned as the amount of enzyme that released 0.1 µmol GAMG equivalent per minute.

Determination of product and yield
Two grams of solid-state medium was sampled, ground by liquid nitrogen, and dissolved in 15 mL water.
The suspension was centrifuged at 4 °C and 10 000 × g, and the supernatant was collected and measured with a metered volume of 100 mL for sample preparation. Then, GAMG and GA were determined by UPLC. UPLC analysis was performed on an ACQUITY UPLC (Waters, USA) instrument using a C18 column (4.6 mm×250 mm, 5 µm, InertSustain, Japan) and UV detector, at the detection wavelength of 254 nm, injection volume of 10 µL, ow rate of 1.0 mL/min, and mobile phase MeOH-0.5% acetate (80:20).
The concentration of GAMG in samples (de ned as C s (mg/mL)) was determined by UPLC with a standard curve. The concentration of GAMG per gram of solid-state medium (de ned as C ssf (mg/g)) was detected by C ssf = (C s × 100)/2. The production of GAMG by SSF (de ned as Y (mg/g)) was quanti ed by Y = C ssf × m/m 0 , whereas m 0 is the weight (g) of initial substrate (licorice straw, 15 g in this study), and m is the weight (g) of solid-state medium after fermentation. The productivity of GAMG by SSF (de ned as P (mg/g/day)) was shown by P = Y/day.

Results And Discussion
3.1 Production of GAMG by C. globosum DX-THS3 using licorice straw as substrate C. globosum DX-THS3 is an endophytic fungus, and it was isolated from Dongxiang wild rice in our previous work (Wang et al., 2015b). In this study, strain DX-THS3 can utilize licorice straw as a substrate for producing GAMG. Strain DX-THS3 was cultivated in licorice straw to produce GAMG by SSF (Fig. 1A). After 20 days of cultivation, C. globosum DX-THS3 covered almost all of the licorice straw (Fig. 1A, right). Solid-state medium was sampled, and the product was analyzed by TLC to detect the GAMG. As shown in Fig. 1B, only GL was detected in the licorice straw (b), but after 20 days of fermentation, considerable GAMG was detected using TLC (c). UPLC was performed for identi cation and further con rmation of these products. Our results con rmed that GAMG was the product in SSF (Fig. 1C). Thus, our results show that GL of licorice straw can be bio-transformed to produce GAMG using licorice straw as a substrate by C. globosum DX-THS3.
GAMG is an innovative functional sweetener with higher sweetness and stronger pharmacological activity than GL (Lin et al., 2009;Li et al., 2017;Mizutani et al., 1994). Biocatalysis of GAMG is a more environment-friendly and e cient than the chemical method (Brieskorn & Lang, 1978). To date, several microorganisms (mainly fungi) are screened for GL transformation to produce GAMG. However, a limited number of microorganisms have a GL-hydrolyzing ability. Especially, microorganisms that can selectively transform GL to generate GAMG are rarely found. The previously reported microorganisms with GLhydrolyzing ability mainly include fungi, such as the lamentous fungus P. purpurogenum Li-3 (Zou et al., 2013) and Talaromyces pinophilus Li-93 , and screened from soil. However, the poor substrate speci city and low e ciency are the main disadvantages that limit the further application of these reported fungi. On the other hand, the production of GAMG by microorganic enzymes (GUS) involve tedious steps, including strain cultivation, fermentation, enzyme extraction, enzymatic reaction, and product separation, that seriously limit the application of microorganic enzymes in industries. Thus, screening microorganism from other sources and a novel strategy for high-e cient utilization of microorganic enzymes should be suggested. In this study, an endophytic fungi C. globosum DX-THS3 selectively and e ciently transformed GL to produce GAMG when using licorice straw as substrate in SSF. To our knowledge, this work is the rst to report the GL production by SSF using endophytic fungi. This research also contributes to the application of endophytic fungi as potential industrial strains in food, biopharmaceutical, and biotechnological industries.
3.2 Optimization of SSF conditions for GAMG production by C. globosum DX-THS3 using licorice straw as substrate We optimized the SSF conditions for GAMG production by C. globosum DX-THS3 using licorice straw as substrate in the present study. The particle size of the substrate is one of the key factors in SSF. Thus, we rst investigated the effect of particle size of licorice straw on GAMG production by C. globosum DX-THS3. As shown in Fig. 2A, 4.56 mg/g (yield, Y) GAMG was produced by C. globosum DX-THS3 when using small-sized licorice straw (0-0.25 mm) as the substrate after 20 days of fermentation; 10.47 mg/g (Y) of GAMG was obtained using a medium particle size (0.26-0.85 mm) of licorice straw after 20 days of fermentation, and 10.35 mg/g (Y) GAMG was detected using licorice straw with a large particle size (0.86-3.0 mm). Our results show that the medium particle size of licorice straw was better than the other particle sizes for the production of GAMG by C. globosum DX-THS3. Furthermore, other fermentation conditions, including temperature, seed age, inoculum size, and solid-liquid ratio (substrate:water, m/v), were optimized. Our results show that 28 ℃ (Y = 10.47 mg/g, Fig. 2B), 96 h seed age (Y = 13.46 mg/g, Fig. 2C), 20% inoculum size (Y = 19.78 mg/g, v/w, Fig. 2D), and 1:3 solid-liquid ratio (Y = 11.75 mg/g, Fig. 2E) were the optimal conditions for the production of GAMG by SSF using C. globosum DX-THS3. These optimal SSF conditions (0.26-0.85 mm licorice straw, 28 °C, 96 h seed age, 20% inoculum size, and 1:3 solid-liquid ratio) were con rmed.
The fermentation conditions can markedly in uence the products (not only the kinds but also the yields) when fermenting using microorganisms (Singh et al., 2017). Similar to liquid fermentation, temperature and seed age should be considered when using microorganisms (most of which belong to fungi) for SSF. However, special fermentation conditions, such as the particle size of substrate and water content, should be primarily considered. In general, oversized or extremely small particle size of substrate is unsuitable for SSF using fungi. Oversized particles can affect substrate release, especially that of lignocellulose, and signi cantly reduce the contact area of fungi with the substrate (Pandey, 2003). On the other hand, an extremely small particle size can in uence fungal growth (Pandey, 2003). Water and substrate powder will mix to form a tight bulk or pellet, which not only signi cantly reduces oxygen transfer but also obstructs in-depth growth of fungal mycelium. In this study, the particle size of licorice straw was rst optimized, and our results showed a suitable particle size of 0.26-0.85 mm ( Fig. 2A) for the production of GAMG by SSF using C. globosum DX-THS3. Water content of substrate is another important factor for SSF by fungi. Low water content is a poor condition for fungal growth. However, a high water content can also inhibit fungal growth, that is, when oxygen transfer is obstructed by high water content of substrate. Thus, suitable water content is required in SSF, and a similar result was obtained in our study (Fig. 2E).

High activity of lignocellulosic enzymes during the degradation of licorice straw by C. globosum DX-THS3
C. globosum DX-THS3 was rst cultured in pre-treated licorice straw under the above fermentation conditions to further investigate the feasibility of this novel strategy for GAMG production. C. globosum DX-THS3 mycelium was grown slowly in the early stage of fermentation (0-6 days). Then, the growth of C. globosum DX-THS3 gradually hastened in the middle stage of fermentation (7-12 days), and the mycelium covered the licorice straw after 20 days of fermentation. Then, deep fermentation was performed (Fig. 3A). The yields of GAMG by C. globosum DX-THS3 using licorice straw as substrate were analyzed during SSF to detect the production of GAMG by C. globosum DX-THS3. The production of GAMG was extremely low in the early and middle stages (0-18 days) of SFF (Fig. 3B). Until 18 days of fermentation, the yield of GAMG signi cantly increased. Our results demonstrate that the yield of GAMG reached 13.73 mg/g after 20 days, and the percent conversion of GL reached 90% (Y = 31.5 mg/g) after about 33 days of SSF (Fig. 3B). Furthermore, the total and reducing sugars were detected during SSF to investigate the utilization of carbon source by C. globosum DX-THS3. The total sugar of substrate continuously decreased during 0-30 days of SSF and stabilized afterward (Fig. 3C). Thus, the growth period of C. globosum DX-THS3 was mainly at 0-30 days of SSF. The reducing sugar increased rapidly at the early stage of SSF (0-7 days), and the highest concentration was detected at 7 days. Then, the reducing sugar was largely utilized by C. globosum DX-THS3 (Fig. 3C). The reducing sugar lowly increased again at the middle stage of SSF (20-22 days, Fig. 3C). The corresponding enzymatic activities, including those of CMCase, FPase, β-glucosidase, and xylanase, were analyzed during SSF to investigate the variation in reducing sugar during SSF by C. globosum DX-THS3. Lignocellulosic enzymatic activities were observed at the early stage of SSF, and the highest activities of CMCase (29.25 U/g), FPase (232.5 U/g), and xylanase (72.52 U/g) were detected at 10, 7, and 7 days of SSF, respectively (Table 1 and Fig. 3D). Meanwhile, our results showed rare β-glucosidase activity during SSF of C. globosum DX-THS3, whereas 6.42 U/g enzymatic activity was detected at 22 days of SSF. Biotransformation has more potential than chemical approaches because of its high yield, high selectivity, and environmental compatibility. Compared with liquid fermentation, SSF has many advantages, including low cost and simple processing, for the production of certain compounds. Meanwhile, excellent lignocellulose-degrading activities for utilization of complex substrates (usually crop straw) by microorganisms are needed. Thus, fungi that harbor rich genes coding lignocellulosic enzymes and excellent lignocellulose-degrading activities are usually used in SSF for the production of  (Table 1 and Fig. 3D) using C. globosum DX-THS3 ( Table 2). The 234.6 U/g FPase activity was detected during SSF, and this value is substantially higher than the other reported FPase activities in SSF using fungi. T. asperellum UC1 (Ezeilo et al., 2019) showed a relative high FPase (26.02 U/g) activity when using oil palm frond leaves as substrate for SSF, and most FPase activity was detected at 0.09-5 U/g ( Table 2). High FPase activity indicates that complex cellulose can be degraded to generate easily hydrolyzed cello-oligomers, which are then further utilized by fungi. Furthermore, compared with the results of other studies, lignocellulose-degrading enzymes, including CMCase, xylanase, and β-glucosidase, from C. globosum DX-THS3 during SSF exhibited relative better enzymatic activities (Tables 1 and 2). Thus, high FPase activity and relative excellent CMCase, xylanase, and βglucosidase activities were detected during SSF by C. globosum DX-THS3, demonstrating their wide application in SSF. Our results also strongly consider C. globosum DX-THS3 as a potential producer for producing lignocellulose-degrading enzymes.

Fructose can signi cantly improve GAMG production by C. globosum DX-THS3
Nitrogen and carbon sources play key roles for production of speci c products by fermentation using microorganism. Additional nitrogen and carbon sources were added to the licorice straw to further increase the productivity of GAMG by using SSF. First, the nitrogen source was optimized for GAMG production by SSF using C. globosum DX-THS3. NH 4 NO 3 , peptone, yeast powder, and yeast extract were used as nitrogen sources for the production of GAMG by C. globosum DX-THS3 (S Fig. 1A). Our results also show that the yield of GAMG was 1.44-(20.21 mg/g) and 1.19-fold (16.79 mg/g) higher than those of the control (14.02 mg/g) when using NH 4 NO 3 and yeast extract as nitrogen sources after 20 days of fermentation, respectively. The GAMG yields were lower than that of the control when using peptone and yeast powder as nitrogen sources after 20 days of fermentation, with values reaching 13.17 and 13.37 mg/g, respectively. We further detected the GUS activity after adding NH 4 NO 3 , peptone, yeast powder, and yeast extract as nitrogen sources to the medium. Our results also showed the higher GUS activity when using NH 4 NO 3 and yeast extract as nitrogen source than the control, whereas those obtained with peptone and yeast powder were lower after 20 days of fermentation (S Fig. 1B). Thus, NH 4 NO 3 as additional nitrogen source can increase the yield of GAMG by C. globosum DX-THS3 in SSF. Meanwhile, the carbon source for GAMG production was investigated (S Fig. 2). The addition of fructose and glucose can produce 18.38 and 17.12 mg/g of GAMG after 20 days of fermentation (S Fig. 2A and 2B), respectively, which denoted increases of 33.9% and 24.7% than those obtained without a carbon source (13.73 mg/g). The addition of sucrose can generate 13.98 mg/g GAMG but showed no signi cant effect on the production of GAMG using C. globosum DX-THS3 (S Fig. 2C). Our results showed the signi cant inhibition of GAMG production when adding lactose to the medium for SSF using C. globosum DX-THS3 after 20 days of fermentation. A total of 9.23 mg/g GAMG was produced, which was a 34% reduction in GAMG compared with that obtained without a carbon source (S Fig. 2D). Furthermore, GUS activities with the addition of carbon sources were further detected after 20 days of SSF. Our results showed similarity to those of GAMG production with carbon source (S Fig. 2E). GUS activity with the addition of fructose was 338 U/g, which was signi cantly higher than that without carbon source (167 U/g). GUS activity with the addition of glucose was 1.5-fold (250 U/g, S Fig. 2F) higher than that without a carbon source (control, 167 U/g). Compared with the control, the addition of sucrose showed no signi cant effect on GUS activity (155 U/g, S Fig. 2G), but the addition of lactose signi cantly inhibited GUS activity (132 U/g, S Fig. 2H). Thus, NH 4 NO 3 and fructose can signi cantly promote GAMG production of C. globosum DX-THS3 by SSF using licorice straw as a medium.
The yield of GAMG during the SSF period with or without carbon and nitrogen sources was detected to further investigate the production of GAMG using C. globosum DX-THS3. First, we detected the yield of GAMG with or without the addition of carbon source (Fig. 4A). The variation trends of GAMG yields were similar. All the test yields of GAMG slowly increased at the initial stage of SSF (0-15 days), whereas those at the middle stage rapidly increased (15-30 days). Then, the yields of GAMG stabilized at about 32 mg/g at the late stage of SSF (after 30 days). Although all the test yields of GAMG exhibited no signi cant difference at the later stage of SSF (S Fig. 3), the productivities of GAMG by SSF with different carbon sources or without a carbon source presented signi cant differences. Compared with the control (33 days), the addition of fructose was the fastest for GAMG production, and 25 days of SSF was used to reach 90% conversion (Y = 31.52 mg/g), 28 days for the addition of glucose, and 30 days for the addition of sucrose. The addition of lactose to the medium can inhibit GAMG production by C. globosum DX-THS3 in SSF, requiring 38 days to reach 90% conversion. Meanwhile, the addition of nitrogen source also showed signi cant effect on the production of GAMG by SSF using C. globosum DX-THS3. As shown in Fig. 4B, compared with that without nitrogen source, the addition of NH 4 NO 3 to the medium achieved improved production of GAMG by C. globosum DX-THS3, with a reduction of 9 days in the production period to transform 90% of GL compared with that without a nitrogen source. Yeast extract showed no effect on the production of GAMG, whereas yeast powder and peptone can inhibit GAMG production. Thus, NH 4 NO 3 and fructose were further optimized. First, different concentrations of NH 4 NO 3 and fructose (3, 5, 7, 9, and 11 mg/g) were added to the SSF medium. Then, GAMG production was detected after 20 days of SSF. Our results show that 7 mg/g NH 4 NO 3 and 5 mg/g fructose were the optimum conditions for GAMG production by C. globosum DX-THS3 using SSF ( Fig. 4C and 4D, respectively). Based on the above results, GAMG was produced faster under these optimal conditions than the control: 0.26-0.85 mm particle size, 28 ℃, 96 h seed age, 20% inoculum size, 1:3 solid-liquid ratio, 7 mg/g NH 4 NO 3 , and 5 mg/g fructose (Fig. 4E). Under these optimal conditions, the percent conversion of GL reached 90% within 15 days, whereas the control needed 35 days, that is, an additional 20 days needed for 90% conversion (Table 1 and Fig. 4E). The productivity of optimization (P = 2.1 mg•g − 1 •day − 1 ) increased by 133.33% compared with that in non-optimized conditions (P = 0.9 mg•g − 1 •day − 1 ).
Microorganic fermentation can be classi ed into three stages: early, middle, and late stages. In general, low-cost and easily available substrates, such as crop straw, are usually used as medium for SSF using fungi. However, complex and adequate enzymes are needed for the degradation of polysaccharides in these substrates (Prajapati et  . Compared with liquid fermentation, more time is needed for fungal cell growth to secrete a complex enzyme system in SSF. Thus, fast accumulation of biomass is the key for shortening the early stage period and increasing the productivity of SSF. To reduce this period, we rst optimized the fermentation conditions, including the particle size, temperature, seed age, inoculum size. and water content. However, the early stage of GAMG production was long (about 25 days) (Fig. 3B). Thus, several nitrogen sources that are easily utilized by microorganisms were considered. Glucose, fructose, and several monosaccharides (popular carbon sources) can be directly entered into glycolysis and TCA cycle for fungal growth (Fig. 5A). Therefore, we considered adding these carbon sources to the medium to increase productivity when using microorganisms for SSF. Our results demonstrate that the early stage period of SSF was reduced by about 10 days when adding fructose and NH 4 NO 3 to the medium, thus signi cantly increasing productivity (Table 1 and Fig. 4E). In this study, lignocellulose and GL of licorice straw were used as carbon sources for C. globosum DX-THS3 growth, and high lignocellulose-degrading enzymatic activities were detected during 5-15 days of fermentation (Fig. 3D). High GUS activities were observed at 20-25 days (Fig. 3C), and the concentration of reducing sugar abruptly increased at 20 days of fermentation. These ndings demonstrate that lignocellulose was rst utilized, followed by GL, by C. globosum DX-THS3. Thus, certain carbon sources were added to the medium to reduce the early stage period and increase the GAMG production. In summary, at the initial period of SSF, several lignocellulosedegrading enzymes were secreted because of the rare biomass of C. globosum DX-THS3. C. globosum DX-THS3 slowly grew. Thus, a long period was needed for the accumulation of C. globosum DX-THS3 to secrete su cient lignocellulose-degrading enzymes. Subsequently, GUS was rapidly secreted by C. globosum DX-THS3 for the utilization of GL as a carbon source to generate GAMG and glucuronic acid (Gur). If several popular carbon sources, such as fructose, are added to medium, C. globosum DX-THS3 will grow fast, which can promote the secretion of lignocellulose-degrading enzymes and fast utilization of lignocellulose, signi cantly reducing the time for production of GAMG (Fig. 5B). Thus, our study provides a novel, fast, and low-cost method for the production of GAMG.

Conclusions
In the present study, C. globosum DX-THS3 can use licorice straw as a medium for SSF. After optimization of fermentation conditions, 90% GL was transformed to GAMG within 15 days, whereas the control needed 35 days. The production of GAMG can reach 2.1 mg•g − 1 •day − 1 , which was 2.33-fold higher than that of the control. Meanwhile, high lignocellulose-degrading enzymatic activities were also detected during SSF. Therefore, our study contributes to the application of SSF in the production of GAMG and strongly demonstrated C. globosum DX-THS3 as a potential candidate for producing lignocellulose-degrading enzymes.