Production of Minor Ginsenosides from Panax notoginseng Flowers by Cladosporium xylophilum

Panax notoginseng flowers have the highest content of saponins compared to the other parts of Panax notoginseng, but minor ginsenosides have higher pharmacological activity than the main natural ginsenosides. Therefore, this study focused on the transformation of the main ginsenosides in Panax notoginseng flowers to minor ginsenosides using the fungus of Cladosporium xylophilum isolated from soil. The main ginsenosides Rb1, Rb2, Rb3, and Rc and the notoginsenoside Fa in Panax notoginseng flowers were transformed into the ginsenosides F2 and Rd2, the notoginsenosides Fd and Fe, and the ginsenoside R7; the conversion rates were 100, 100, 100, 88.5, and 100%, respectively. The transformation products were studied by TLC, HPLC, and MS analyses, and the biotransformation pathways of the major ginsenosides were proposed. In addition, the purified enzyme of the fungus was prepared with the molecular weight of 66.4 kDa. The transformation of the monomer ginsenosides by the crude enzyme is consistent with that by the fungus. Additionally, three saponins were isolated from the transformation products and identified as the ginsenoside Rd2 and the notoginsenosides Fe and Fd by NMR and MS analyses. This study provided a unique and powerful microbial strain for efficiently transformating major ginsenosides in P. notoginseng flowers to minor ginsenosides, which will help raise the functional and economic value of the P. notoginseng flower.


Introduction
Panax notoginseng (Burk.) F.H. Chen (Araliaceae) is a traditional precious Chinese herbal medicine, which mainly grows in the Yunnan and Guangxi provinces in southwest China. Saponins are the main bioactive ingredients in different parts of P. notoginseng. The part of the P. notoginseng flower (PNF) contains more than 20% of the total saponins, which is the highest saponin content in the whole plant [1][2][3]. The ginsenosides Rb 1 , Rb 2 , Rb 3 , and Rc and the notoginsenosides Fa and Fc are the major saponins in the PNF and belong to the protopanaxadiol (PPD) ginsenosides, but minor ginsenosides with minimal levels have higher pharmacological activity than the major natural ginsenosides. Studies have shown that minor saponins containing less sugar may show higher bioavailability, better cell permeability, and other advantages; so, minor saponins show higher pharmacological activity [4][5][6]. For example, ginsenoside Rd 2 can prevent or treat thrombotic diseases; notoginsenoside Fe can treat cardiovascular and cerebrovascular diseases and inhibit diet-induced obesity [7,8]. The minor ginsenosides have similar structures to the major ginsenosides and can be transformed from the major ginsenosides. Therefore, we can prepare minor ginsenosides from major ginsenosides in PNF. At present, the main methods of obtaining minor ginsenosides include physical transformation, chemical transformation, biological transformation and cloned ginsenoside enzyme transformation [9].

Ginsenoside-Transforming Activity Screening and Characterization of Strain S7
PPD ginsenosides are the main components of PNF; among them, ginsenoside Rb 1 is one of the major saponins in PNF and a representative of the protopanaxadiol (PPD) ginsenosides.
We screened six strains for ginsenoside Rb 1 transformation activity by the TLC methods. The results showed that the strains S7, S3, and S17 have less polar spots on the TLC, which indicated that the three strains have the ability to transform ginsenoside Rb 1 into another saponin. Compared with strains S3 and S17, strain S7 has a higher transformation rate; there was almost no spot of substrate on the TLC, indicating that the transformation substrate was almost exhausted. In addition to this, the main product of ginsenoside Rb 1 by the strain S3 and S17 was ginsenoside Rd, which is the main component of the flower, not the target rare saponin, while the transformation product of strain S7 was the rare ginsenoside, with no intermediate product. The TLC analysis of the transformation products by different strains showed that strain S7 exhibited a significant ability to transform Rb 1 compared to the other stains (Supplementary Materials Figure S1). So, strain S7 was selected for the further experiments.
After strain S7 was cultured on PDA medium for 4 days, the following colony characteristics were observed: the surface was olive green and villous and the colony was flat, as shown in Figure S2A. Its morphological characteristics were observed under light microscope as follows: the conidiophores were erect, slightly curved, nodal, septate and slightly branched. The side formed a conidia chain which was branching and light brown.
The conidia morphology was variable and smooth, nearly spherical, elliptic, and long cylindrical, as shown in Figure S2B [17,18]. Based on the sequencing of the ITS rDNA gene and a comparison in the GenBank database, it was found that strain S7 belonged to the genus Cladosporium and exhibited significant similarity to Cladosporium xylophilum in Figure S2C.

Qualitative and Quantitative Analysis of Major Saponins in PNF by HPLC
Using 8 mg PNF extract (marked as m, m = 8 mg), they were dissolved in 1 mL methanol (marked as V t , V t = 1 mL) as the analysis sample. The injection volume was 20 µL. The purpose of the HPLC analysis is to obtain the peak area of each saponin and calculate the contents of each saponin of the major saponins in PNF according to the standard curve (marked as m 1 ).
C: the concentration obtained by plugging the peak area of the major saponins into the standard curve, mg/mL; Vi: the injection volume, 20 µL.
The purpose of analyzing the major saponins in the PNF is to calculate the conversion rate of those saponins during the biotransformation process by C. xylophilum. The qualitative and quantitative analyses of the major saponins in the PNF by the HPLC method are shown in Figure 1. The results showed that the contents of the major saponins (notoginsenosides Fa and Fc and ginsenosides Rb 1 , Rb 2 , Rb 3 , Rd, and Rc) in the PNF were 2.80, 0.29, 0.60, 0.52, 4.80, 0.15, and 2.40%, respectively. 3 acteristics were observed: the surface was olive green and villous and the colony was flat, 98 as shown in Figure S2A. Its morphological characteristics were observed under light mi-99 croscope as follows: the conidiophores were erect, slightly curved, nodal, septate and 100 slightly branched. The side formed a conidia chain which was branching and light brown. 101 The conidia morphology was variable and smooth, nearly spherical, elliptic, and long cy-102 lindrical, as shown in Figure S2B [17,18]. Based on the sequencing of the ITS rDNA gene 103 and a comparison in the GenBank database, it was found that strain S7 belonged to the 104 genus Cladosporium and exhibited significant similarity to Cladosporium xylophilum in Fig-105 ure S2C. 106

107
Using 8 mg PNF extract (marked as m, m = 8 mg), they were dissolved in 1mL meth-108 anol (marked as Vt, Vt = 1mL) as the analysis sample. The injection volume was 20 µL. The 109 purpose of the HPLC analysis is to obtain the peak area of each saponin and calculate the 110 contents of each saponin of the major saponins in PNF according to the standard curve 111 (marked as m1).
C: the concentration obtained by plugging the peak area of the major saponins into the 113 standard curve, mg/mL; Vi: the injection volume, 20 µL. 114 The purpose of analyzing the major saponins in the PNF is to calculate the conversion 115 rate of those saponins during the biotransformation process by C. xylophilum. The quali-116 tative and quantitative analyses of the major saponins in the PNF by the HPLC method 117 are shown in Figure 1. The results showed that the contents of the major saponins (noto-118 ginsenosides Fa and Fc and ginsenosides Rb1, Rb2, Rb3, Rd, and Rc) in the PNF were 2.80, 119 0.29, 0.60, 0.52, 4.80, 0.15, and 2.40%, respectively.

HPLC Analysis the Dynamic Change of Major Saponins in PNF Transformed by C. xylophilum
During the biotransformation process of the major saponin in the PNF by C. xylophilum, it was regularly monitored by HPLC analysis ( Figure 2). As shown in Figure 3, the notoginsenoside Fa and the ginsenosides Rb 1 , Rb 2 , Rc, Rb 3 , and Rd that comprised the major portion of the PNF were rapidly transformed into other saponins in the early stage of the reaction (1-5 days). After 10 days of reaction, the notoginsenoside Fa and the ginsenosides Rb 1 , Rb 2 , Rb 3 , and Rd were completely transformed by C. xylophilum, and During the biotransformation process of the major saponin in the PNF by C. xy-126 lophilum, it was regularly monitored by HPLC analysis ( Figure 2). As shown in Figure 3, 127 the notoginsenoside Fa and the ginsenosides Rb1, Rb2, Rc, Rb3, and Rd that comprised the 128 major portion of the PNF were rapidly transformed into other saponins in the early stage 129 of the reaction (1-5 days). After 10 days of reaction, the notoginsenoside Fa and the gin-130 senosides Rb1, Rb2, Rb3, and Rd were completely transformed by C. xylophilum, and the 131 conversion rate reached 100%. After 15 days of reaction, only notoginsenoside Fc and gin-132 senoside Rc were left in the PNF, and the final conversion rates were 53.4 and 88.5%, re-133 spectively. 134 Conversion rate (%) = total saponins − remaining saponins total saponins × 100%  (3); ginsenoside Rc (4); ginsenoside Rb 2 (5); ginsenoside Rb 3 (6); ginsenoside Rd (7); Gpy17 (8); notoginsenoside Fe (9); ginsenoside Rd 2 (10); notoginsenoside Fd (11); ginsenoside F 2 (12). Major saponins in PNF transformated by C. xylophilum for different days.

HPLC Analysis of the Transformation Pathways of Monomer Ginsenosides Rb 1 , Rb 2 , Rb 3 , Rc, Notoginsenosides Fa and Fc by C. xylophilum
In order to further verify the transformation pathways of the main saponins in the PNF, the ginsenosides Rb 1 , Rb 2 , Rb 3 , and Rc and the notoginsenosides Fa and Fc were used as substrates for the transformation experiments, respectively.
The transformation pathway of ginsenoside Rb 1 is proposed in Figure 4A. The ginsenoside Rb 1 molecule contains four β-glucopyranosyl moieties at the C-3 and C-20 position of aglycone. Based on the results obtained by HPLC analysis ( Figure S3), we can see that there are peaks of small polar products in the product, which were identified as F 2 by comparing their retention time with the standard ginsenoside F 2 ; so, we suggest that Rb 1 was biotransformed into F 2 by C. xylophilum. The biotransformation of Rb 1 into F 2 can occur through pathways of two types, depending on their structures. Firstly, the enzyme from C. xylophilum attacked the outer β-(1→2)-glucosidic linkage to the C-3 position of aglycone to produce Gyp17 from Rb 1 and was then followed by the hydrolysis of the outer β-(1→6)-glucosidic to the C-20 position to produce F 2 from Gyp17. Secondly, the enzyme from C. xylophilum attacked the outer β-(1→6)-glucosidic linkage to the C-20 position of aglycone to produce Rd from Rb 1 and was then followed by the hydrolysis of the outer β-(1→2)-glucosidic to the C-3 position to produce F 2 from Rd. In order to further verify the transformation pathways of the main saponins in the 148 PNF, the ginsenosides Rb1, Rb2, Rb3, and Rc and the notoginsenosides Fa and Fc were used 149 as substrates for the transformation experiments, respectively. 150 The transformation pathway of ginsenoside Rb1 is proposed in Figure 4A. The gin-151 senoside Rb1 molecule contains four β-glucopyranosyl moieties at the C-3 and C-20 posi-152 tion of aglycone. Based on the results obtained by HPLC analysis (Figure S3), we can see 153 that there are peaks of small polar products in the product, which were identified as F2 by 154 comparing their retention time with the standard ginsenoside F2; so, we suggest that Rb1 155 was biotransformed into F2 by C. xylophilum. The biotransformation of Rb1 into F2 can oc-156 cur through pathways of two types, depending on their structures. Firstly, the enzyme 157 from C. xylophilum attacked the outer β-(1→2)-glucosidic linkage to the C-3 position of 158 aglycone to produce Gyp17 from Rb1 and was then followed by the hydrolysis of the outer 159 β-(1→6)-glucosidic to the C-20 position to produce F2 from Gyp17. Secondly, the enzyme 160 from C. xylophilum attacked the outer β-(1→6)-glucosidic linkage to the C-20 position of 161 aglycone to produce Rd from Rb1 and was then followed by the hydrolysis of the outer β-162 (1→2)-glucosidic to the C-3 position to produce F2 from Rd. 163 The transformation pathway of notoginsenoside Fa is proposed in Figure 4B. The 164 notoginsenoside Fa contains one α-(1→2)-xylopyranosyl (outer) and two β-glucopyra-165 nosyl moieties (inner) at the C-3 position, with two β-glucopyranosyl moieties at the C-20 166 The transformation pathway of notoginsenoside Fa is proposed in Figure 4B. The notoginsenoside Fa contains one α-(1→2)-xylopyranosyl (outer) and two β-glucopyranosyl moieties (inner) at the C-3 position, with two β-glucopyranosyl moieties at the C-20 position of aglycone. Based on the results obtained by the HPLC analysis ( Figure S3), we can see that there is a main peak of small polar products in the HPLC spectrum; so, we suggest that Fa can be transformed into another ginsenoside by C. xylophilum. The product's molecular formula of C 53 H 90 O 22 was determined by HR-ESI-MS at m/z 1077.5843 [M-H]-(calcd. for 1077.5845). Ginsenoside R 7 has the same molecular formula of C 53 H 90 O 22 . Due to the existence of isomers, we analyzed the possible compounds with the same molecular formula in Panax plants. According to the characteristics of the saponin transformation pathway (which usually hydrolyzes one or more glycosyl fragments), we determined that the product of substrate (ginsenoside Fa) transformed by C. xylophilum was ginsenoside R 7 .
The enzyme from C. xylophilum attacked the outer β-(1→6)-glucosidic linkage to the C-20 position of aglycone to produce R 7 from Fa. In addition to the HPLC analysis, the HR-ESI-MS analysis of the transformation product of notoginsenoside Fa was further verification that the product was ginsenoside R 7 , as shown in Figure S4.
The transformation pathway of ginsenoside Rb 2 is proposed in Figure 4C. The ginsenoside Rb 2 molecule contains one α-(1→6)-arabinopyranosyl (outer) and one β-glucopyranosyl moiety (inner) at the C-20 position, with two β-glucopyranosyl moieties at the C-3 position of aglycone. Based on the results obtained by the HPLC analysis ( Figure S3), we can see that there are peaks of small polar products in the product, which were identified as Rd 2 by comparing their retention times with standard ginsenoside Rd 2 ; so, we suggested that Rb 2 Molecules 2022, 27, 6615 6 of 12 was transformed into ginsenoside Rd 2 by C. xylophilum. The enzyme from C. xylophilum attacked the outer β-(1→2)-glucosidic linkage to the C-3 position of aglycone to produce Rd 2 from Rb 2 . Similarly, the enzyme from C. xylophilum attacked the outer β-(1→2)-glucosidic linkage to the C-3 position of aglycone to produce Fe from Rc ( Figure 4D and Figure S3). The enzyme from C. xylophilum attacked the outer β-(1→2)-glucosidic linkage to the C-3 position of aglycone to produce Fd from Rb 3 ( Figure 4E and Figure S3). 6 aglycone to produce Rd2 from Rb2. Similarly, the enzyme from C. xylophilum attacked the 189 outer β-(1→2)-glucosidic linkage to the C-3 position of aglycone to produce Fe from Rc 190 ( Figure 4D, S3). The enzyme from C. xylophilum attacked the outer β-(1→2)-glucosidic 191 linkage to the C-3 position of aglycone to produce Fd from Rb3 ( Figure 4E, S3). 192 The enzyme of C. xylophilum can hydrolyze lateral glucose at the C-20 and C-3 of 193 ginsenoside Rb1 to F2 through two pathways. In addition to this, the enzyme can hydrolyze 194 lateral glucose at the C-20 or C-3 of notoginsenoside Fa and the ginsenosides Rb2, Rb3, and 195 Rc through a single pathway, but cannot hydrolyze the arabinose, xylose, and inside glu-196 cose. It indicated that the enzyme from this strain was highly specific, and it could trans-197 form different saponins into specific ginsenosides. 198 The maximal concentration of minor ginsenosides in the transformation products of 199 the major saponins in the PNF by using C. xylophilum occurred on the 10th day, as is 200 shown in Figure 5. The contents of the minor ginsenosides F2 and Rd2 and the noto-201 ginsenosides Fd and Fe were 0.99, 0.67, 0.24, and 0.24 mg/mL, respectively. The enzyme of C. xylophilum can hydrolyze lateral glucose at the C-20 and C-3 of ginsenoside Rb 1 to F 2 through two pathways. In addition to this, the enzyme can hydrolyze lateral glucose at the C-20 or C-3 of notoginsenoside Fa and the ginsenosides Rb 2 , Rb 3 , and Rc through a single pathway, but cannot hydrolyze the arabinose, xylose, and inside glucose. It indicated that the enzyme from this strain was highly specific, and it could transform different saponins into specific ginsenosides.
The maximal concentration of minor ginsenosides in the transformation products of the major saponins in the PNF by using C. xylophilum occurred on the 10th day, as is shown in Figure 5. The contents of the minor ginsenosides F 2 and Rd 2 and the notoginsenosides Fd and Fe were 0.99, 0.67, 0.24, and 0.24 mg/mL, respectively.  213 The results of the SDS-PAGE showed that the purified enzyme was a single band, 214 and its molecular weight was estimated to be 66.4 kDa according to the relative migration 215 distance of the molecular weight markers in electrophoresis ( Figure S5). The molecular 216 weight of the protein was similar to that reported in the literature [19][20][21]. The β-gluco-217 sidase activity from C. xylophilum is 129 U/mL for pNP-β-D-glucopyranoside (as a dry 218 weight base). 219

220
The results of the crude enzyme transformation were consistent with those of C. xy-221 lophilum ( Figure S6). The biotransformation of the monomer saponins by the crude en-222 zymes was studied in the pH range of 4 to 8 and the temperature range of 30 to 70 °C 223 ( Figure 6). The optimal pH for the transformation of the ginsenosides was in the range of 224 5-6. These results suggest that the biotransformation of ginsenosides by crude enzymes 225 was more desirable in weak acidic conditions (pH 5-6) rather than in neutral and basic 226 conditions. The optimal temperature was 50 °C for the biotransformation of the ginseno-227 sides by crude enzymes. 228 Figure 5. The contents change of ginsenosides F 2 and Rd 2 and notoginsenosides Fd and Fe from the transformation products of ginsenosides Rb 1 , Rb 2 , Rb 3 , and Rc by C. xylophilum.

Enzyme Purification and Characterization from C. xylophilum
The results of the SDS-PAGE showed that the purified enzyme was a single band, and its molecular weight was estimated to be 66.4 kDa according to the relative migration distance of the molecular weight markers in electrophoresis ( Figure S5). The molecular weight of the protein was similar to that reported in the literature [19][20][21]. The β-glucosidase activity from C. xylophilum is 129 U/mL for pNP-β-D-glucopyranoside (as a dry weight base).

Characterization of the Crude Enzymes for Monomer Saponins Transformation
The results of the crude enzyme transformation were consistent with those of C. xylophilum ( Figure S6). The biotransformation of the monomer saponins by the crude enzymes was studied in the pH range of 4 to 8 and the temperature range of 30 to 70 • C ( Figure 6). The optimal pH for the transformation of the ginsenosides was in the range of 5-6. These results suggest that the biotransformation of ginsenosides by crude enzymes was more desirable in weak acidic conditions (pH 5-6) rather than in neutral and basic conditions. The optimal temperature was 50 • C for the biotransformation of the ginsenosides by crude enzymes.   Figure S7). The compound showed identical NMR signals (Table S1) to those described in the literature [22,23].  Figure S8). The compound showed identical NMR signals (Table S1) to those described in the literature [24,25].  Figure S9). The compound showed identical NMR signals (Table S1) to those described in the literature [26,27].   Figure S7). The compound showed identical NMR signals (Table S1) to those described in the literature [22,23].  Figure S8). The compound showed identical NMR signals (Table S1) to those described in the literature [24,25].

Isolation, Screening, and Species Identification of Fungi
Sixteen strains of fungi were isolated by the soil dilution plate method [28]. The isolated and purified strains were cultured on PDA medium, cultured at 26 • C for 3-4 days. The purified strain was stored in a refrigerator at 4 • C for subsequent studies. The strains with high ginsenoside transformation activity were screened through the transformation activity of ginsenoside Rb 1 . The amplification and sequencing of the ITS rDNA gene was completed by the Kunming Branch of Tsingke Biotechnology Co., Ltd (Branch of Tsingke Biotechnology Co., Ltd., Kunming, China). The isolated strain S7 was identified through morphological observation, biochemical characteristics, and phylogenetic analysis.

Preparation of Saponins in PNF
In this experiment, the PNF were extracted by the ethanol reflux extraction method. Sixty percent ethanol was used as the extraction solution; the liquid-solid ratio was 1:14; and the water bath at 60 • C was refluxed for 1.5 h, twice. The final extract yield was 40%. The extract was treated with D101 macroporous adsorption resin.

Biotransformation of Saponins in PNF by C. xylophilum
The biotransformation procedure was performed using PDB medium with 0.4 mg/mL saponins in PNF in a shaking incubator (160 rpm) at 26 • C for 15 days. Samples were withdrawn at regular intervals during fermentation (1, 5, 7, 10, 13, 15 d).

Biotransformation of Monomer Ginsenosides Rb 1 , Rb 2 , Rb 3 , Rc and Notoginsenosides Fa and Fc by C. xylophilum
The biotransformation procedure was performed using PDB medium with 0.05 mg/mL of the ginsenosides Rb 1 , Rb 2 , Rb 3 , and Rc and the notoginsenosides Fa and Fc in a shaking incubator (160 rpm) at 26 • C for 10 days.

Preparation of Crude Enzyme
The culture medium was filtered with four layers of gauze to remove mycelia, and the supernatant was collected; When ammonium sulfate was added into the supernatant and the saturation reached 75%, the supernatant was precipitated for 1 h, then centrifuged (4000 r/min) for 20 min; the supernatant was discarded and the precipitation dissolved in HAc-NaAc (pH 5.0) buffer. The solution was centrifuged again (4000 r/min) for 20 min to remove the insoluble hybrid proteins. The crude enzyme solution was freeze-dried after dialysis for 24 h in HAc-NaAc buffer (pH 5.0).

Purification of Crude Enzyme
The crude enzyme was purified by anion exchange column DEAE cellulose DE-52 (φ1.5 cm × 15 cm). The enzymatic activity of hydrolyzed ginsenoside Rb 1 was detected, and the part of the hydrolyzed ginsenoside Rb 1 was collected and then lyophilized. The purified protein was determined by Polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime biotechnology, Shanghai, China).

β-glucosidase Activity Determination
Using pNP-β-D-glucopyranoside (pNPG) as a substrate, the activity of β-glucosidase was detected by colorimetry. The activity unit of β-glucosidase was defined as the amount of enzyme required for the hydrolysis of 1 mL enzyme solution for 1 min to produce 1 µmol p-nitrophenol (pNP).

Biotransformation of Monomer Saponins by Crude Enzymes
The biotransformation procedure was performed as follows: dissolve the monomer saponins (ginsenosides Rb 1 , Rb 2 , Rb 3 , and Rc and notoginsenosides Fa and Fc) in 1 mL of pH HAc-NaAc buffer (pH 5.0) and mix with the same volume of crude enzyme; incubate at 50 • C for 2 days (the final substrate concentrations of the monomer ginsenosides were 0.05 mg/mL). In addition, the biotransformation of the monomer saponins by crude enzymes was studied in the pH range of 4 to 8 and the temperature range of 30 to 70 • C.

Preparation of Notoginsenoside Fe, Ginsenoside Rd 2 , and Notoginsenoside Fd from Main Saponins in PNF Transformed by C. xylophilum
The biotransformation procedure was performed using PDB medium with 0.4 mg/mL of saponins in PNF in a shaking incubator (160 rpm) at 26 • C for 15 days. The main saponins in PNF were transformed into minor ginsenosides by C. xylophilum. The cultivation of liquid was extracted with n-butanol 3 times, and the extract was concentrated under reduced pressure to obtain 21 g residue. The extract was eluted by D101 macroporous resin column chromatography with a gradient elution of an ethanol-water solvent system to obtain four fractions Fr. A~D. Fr. B was separated by repeated silica gel column chromatography (CH 2 Cl 2 -MeOH, 10:1~6:1) to obtain compound 1 (13.3 mg). Fr. C was separated by repeated silica gel column chromatography (CH 2 Cl 2 -MeOH, 10:1~5:1) to obtain compound 2 (35 mg) and compound 3 (17 mg).

Conclusions
This was the first report of the unique saponin conversion activities of C. xylophilum.
Our study suggests that this fungus can convert the main saponins in the PNF to minor ginsenosides. When the monomer saponin is used as the transformation substrate, the transformation rate is high, and the transformation product is specific. Therefore, the fungus can specifically transform the main saponins in the PNF to produce minor ginsenosides, with a single transformation product and few by-products.
When the biotransformation of saponins in PNF (mainly including: ginsenosides Rb 1 , Rb 2 , Rb 3 , and Rc and notoginsenosides Fa and Fc) by C. xylophilum, the content of Fc was significantly reduced. However, when there was the biotransformation of the monomer notoginsenoside Fc by C. xylophilum, the Fc was not transformed. It was speculated that a promotion effect was produced between the saponins during the transformation of the main saponins in PNF by C. xylophilum. When Gpy17 was produced in the product, the transformation effect of notoginsenoside Fc was more obvious ( Figure S10). This conjecture mainly refers to the research in this literature [29], and the combination of different substrates can be used for selective biotransformation.
We found that C. xylophilum isolated from P. notoginseng soil was highly effective and selective in the biotransformation of the main saponin (the notoginsenosides Fa and Fc and the ginsenosides Rb 1 , Rb 2 , Rc, and Rb 3 ) in the PNF into minor saponins. The conversion rate was 100%, except for ginsenoside Rc at 88.5% and notoginsenoside Fc at 55.3%. The results of the present study suggest that C. xylophilum can be used to produce valuable minor ginsenosides from the main saponin in the PNF, with high biotransformation efficiency. These findings will lay a solid foundation for the construction of genetically engineered strains and eventually the large-scale preparation of minor saponins.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules27196615/s1, Table S1: 13 C NMR data of compounds 1-3 (notoginsenosides Fd, Fe and ginsenoside Rd 2 ) in C 5 D 5 N, Figure S1: TLC analysis of the transformation of ginsenoside Rb 1 by different strains, Figure S2: morphology and ITS gene identification of strain S7, Figure S3: HPLC analysis of the transformation products of monomer ginsenosides Rb 1 , Rb 2 , Rb 3 , Rc, notoginsenosides Fa and Fc by C. xylophilum, Figure S4: MS analysis of transformation products of notoginsenoside Fa by C. xylophilum, Figure S5: SDS-PAGE analysis of the purified β-glucosidase from C. xylophilum after protein staining with Coomassie Brilliant Blue solution, Figure S6: biotransformation of ginsenosides Rb 1 , Rb 2 , Rb 3 , Rc, notoginsenosides Fa and Fc by crude enzymes, Figure S7: 1 H NMR, 13 C NMR (C 5 D 5 N), and MS spectra of compound 1, Figure S8: 1 H NMR, 13 C NMR (C 5 D 5 N), and MS spectra of compound 2, Figure S9: 1 H NMR, 13 C NMR (C 5 D 5 N), and MS spectra of compound 3, Figure S10: HPLC analysis of the transformation products of mixture of same mass of ginsenoside by C. xylophilum.