Effects of mixed carbon sources on galactose and mannose content of exopolysaccharides and related enzyme activities in Ganoderma lucidum

Abstract

This work investigated the effects of mixed carbon sources on the monosaccharide composition of exopolysaccharides (EPS) and activity of related enzymes, phosphoglucose isomerase (PGI), α-phosphoglucomutase (PGM), phosphomannose isomerase (PMI) and GDP-D-Man pyrophosphorylase (GMP), in the EPS biosynthesis by Ganoderma lucidum. Combinations of two carbon sources, glucose (Glc) + mannose (Man) and Glc + galactose (Gal), were applied at various mass ratios in the liquid culture of G. lucidum. The results showed that Glc, Gal, and Man were the major monosaccharides in G. lucidum EPS, and the combined mole percentages of monosaccharides were >80%, the same as those in the carbon source mixture. Activities of PGI, PMI, and GMP were correlated with Man mole percentage in EPSs and were enhanced by increasing the proportion of Gal for the carbon source Glc + Gal. PGM activity was correlated with Gal mole percentage and was reduced by increasing the Man proportion for Glc + Man. The expression of four genes encoding PGI, PMI, GMP and PGM reached peak levels on day 6 of culture, whereas the enzyme activities increased steadily from day 3 to 8. The monosaccharide composition of G. lucidum EPS for various mixed carbon source conditions thus appeared to be controlled by the translational level of genes encoding PGM, PGI, PMI, and GMP. These findings will be helpful to control the monosaccharide composition for desired biological activity of EPS produced by G. lucidum fermentation.

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1. Introduction

Ganoderma lucidum (Lingzhi) is a well-known medicinal mushroom in traditional Chinese medicine which has been used in China and some other oriental countries over thousand years for promoting health and longevity. The extracts of G. lucidum mushroom and fungal mycelium have shown several notable bioactivities such as antitumor, antioxidant, antibacterial, anti-inflammatory, and anti-diabetic properties.^1–3 Polysaccharides are recognized as the major bioactive molecules of G. lucidum and most other medicinal mushrooms, which have antitumor, immunomodulatory and other medicinal properties.^4,5

Liquid or submerged fermentation is an important process in the industry for mass production of mycelial biomass and bioactive products of G. lucidum and other important medicinal mushrooms. Submerged fermentation has been widely explored as a viable process for production of exopolysaccharides (EPS), though it has found few industrial applications due to the low productivity. Manipulation or optimization of the culture conditions and the nutrient media for mycelial fermentation is a common and direct approach for improving the EPS production by G. lucidum.^6,7 The major nutrients and fermentation conditions affect not only the quantity of EPS produced but also the composition and molecular properties of EPS. It is crucial to monitor and control the chemical properties of EPS during the fermentation in order for their reliable application as a nutraceutical and pharmaceutical agents.⁸ Among various nutrients in the culture medium, the carbon source may have a significant influence on the production and composition of EPS. Glucose (Glc) is the most common sugar and carbon nutrient in microbial fermentation for EPS production and also for EPS production in G. lucidum mycelial fermentation.

Previous studies have shown that the sugar components in the culture nutrients are often different from the monosaccharide constituents of the EPS derived from the fungal cultures. In Phellinus linteus culture, Glc percentage in EPS was over 85% under five different carbon sources, including Glc, Gal, Man, arabinose and starch, and thus Glc was the primary monosaccharide in the P. linteus EPS.⁹ In Paecilomyces hepiali cultures, EPS had a high content of Man with Glc as the carbon source and a high content of Glc with Man as the carbon source.¹⁰ However, our previous study showed that the major monosaccharides in EPSs are glucose (Glc), galactose (Gal), and mannose (Man) when G. lucidum is cultured with a single carbon source.¹¹

The biosynthesis pathways for industrially important microbial EPS have been elucidated in certain bacteria based on identification of intermediate compounds and key enzyme activities.^12,13 Little is known about the biosynthesis pathway of G. lucidum EPS and the effects of different carbon sources on the EPS monosaccharide composition. Previous study of our group identified key enzymes that control monosaccharide composition in G. lucidum EPS.¹¹ However, the use of a single carbon source obscured the role of key enzymes in synthesis of each of the three monosaccharides, since the monosaccharide the same as those in the carbon source was excessively present in G. lucidum EPSs. In the present study, we used two carbon sources mixtures containing two of the three monosaccharides (Glc + Man, or Glc + Gal), and studied the synthetic process of the third monosaccharide (Gal, or Man) by comparing monosaccharide composition and related enzyme activities in EPS synthesis.

2. Materials and methods

2.1. G. lucidum submerged fermentation conditions

G. lucidum mycelia was maintained in potato dextrose agar (PDA) slants at 4 °C. Seed medium consisted of 20 g l⁻¹ Glc, 5 g l⁻¹ yeast nitrogen base without amino acids (YNB), 5 g l⁻¹ tryptone, 3 g l⁻¹ KH₂PO₄, and 2 g l⁻¹ MgSO₄·7H₂O, at initial pH 6.0. Fermentation medium consisted of 5 g l⁻¹ tryptone, 3 g l⁻¹ KH₂PO₄, 2 g l⁻¹ MgSO₄·7H₂O, and 20 g l⁻¹ mixed carbon source (Glc + Gal, or Glc + Man, each with ratio 1 : 1 or 1 : 2), at initial pH 6.0. G. lucidum mycelium squares from PDA slants were inoculated into 80 ml seed medium and cultured at 30 °C on a rotary shaker (150 rpm) for 11 days. Seed medium containing G. lucidum pellets was inoculated into 150 ml fermentation medium and cultured under the above conditions for 6 days.

2.2. Monosaccharide composition analysis

Culture broth was collected on days 3, 6, and 8 for each of the four carbon source mixture experimental conditions, and crude EPS was obtained from broth supernatant by adding 75% (v/v) ethanol, standing overnight, and centrifuging. The crude EPS was dissolved in distilled water, and the solution was further centrifuged to remove insoluble substrate. Crude EPS in solution was concentrated by lyophilization, and 20 mg EPS was hydrolyzed with H₂SO₄ (2 ml of 1 mol l⁻¹ solution) and neutralized with BaCO₃. Monosaccharides resulting from the hydrolysis were reacted successively with hydroxylamine hydrochloride, pyridine, and acetic anhydride, and then determined by gas–liquid chromatography with reference to standards. Mole percentage values of monosaccharides were calculated based on content of inositol as internal standard.

2.3. Activity assay of enzymes related to G. lucidum EPS synthesis

G. lucidum mycelium was collected on days 3, 6, and 8 for each of the four carbon source mixture conditions, washed three times with 20 mM phosphate buffer (pH 6.5), frozen by liquid nitrogen, ground, and suspended in phosphate buffer solution. The solution was centrifuged to remove cell debris, and activities of nine enzymes related to EPS synthesis in the cell extract were assayed by spectrophotometry at 30 °C. Aliquots (30 μl) of cell extract were added to initiate the enzyme assay reaction, and enzyme activity was assayed as in our previous study.¹¹ Enzyme activity was calculated using the molar extinction coefficient of NAD(P)H (ε₃₄₀ = 6220 M⁻¹ cm⁻¹), with one activity unit defined as the amount of enzyme required to oxidize 1 nmol NAD(P)H per minute. The blank for each enzyme was run under the same reaction condition but without cell extract.

2.4. RNA and cDNA preparation, and quantitative real-time PCR (qRT-PCR)

G. lucidum mycelium was frozen in liquid nitrogen, ground, and suspended in phosphate buffer solution. Total RNA was extracted using a Trizol Total RNA Purification Kit (Sangon; Shanghai, China), and then reverse transcribed to cDNA using a RevertAid First Strand cDNA Synthesis Kit (Fermentas; Ontario, Canada).

Expression levels of pgm, pgi, pmi1, and pmi2 (encoding enzymes PGM, PGI, and PMI) were analyzed by qRT-PCR using SYBR green (TaKaRa, Japan) according to the manufacturer's instructions. Primers used for qRT-PCR are listed in Table 1. Product specificity was confirmed by melting curve analysis. Gene expression levels were normalized against that of rns, as reported previously.¹⁴

Table 1 Primer sequences for cDNA amplification

mRNA	Primer sequence
pgi	Forward	5′-ACTCGCATCTGAATACGCC-3′
	Reverse	5′-GGATACGGTGGTGCTTGG-3′
pgm	Forward	5′-TTGCCAAGTTCATCATCGG-3′
	Reverse	5′-CCTCCTGCGTGTCCTTATTG-3′
pmi1	Forward	5′-TCAATACATAATCACCGACCAC-3′
	Reverse	5′-AGGAATCATCCTGCCAATC-3′
pmi2	Forward	5′-CTGCTCGCCTTGCACCTAAC-3′
	Reverse	5′-TCTCCGCCTTAGACTTCAGC-3′
Rns	Forward	5′-GAGAAACGAAGGTTAGGGTAGG-3′
	Reverse	5′-CACAAGGCGGAATGGTTATTG-3′

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2.5. Statistical analysis

Data were expressed as mean ± SD, and analyzed using software program GraphPad Prism 5.0. Differences with p < 0.05 were considered significant.

3. Results

3.1. Effects of carbon sources on G. lucidum EPS monosaccharide mole percentage

The carbon sources used for submerged fermentation of G. lucidum were mixtures of Glc + Gal, or Glc + Man, with ratios 1 : 1 or 1 : 2. With these mixed carbon sources, the major monosaccharides of G. lucidum EPSs were Glc, Gal, and Man, and their mole percentages changed between days 3 and 8 of culture (Table 2). The combined mole percentage of EPS monosaccharides the same as those in the carbon sources were >80%, indicating that mole percentages of EPS monosaccharides were determined by the carbon sources. The mole percentage of Glc declined from day 3 to 8 for all carbon source conditions except Glc + Man 1 : 1. Accumulation of sugars in EPSs, which were mixed with Glc in the medium, was observed during the culture period. For all conditions, mole percentage of Gal increased steadily from day 3 to 8, whereas mole percentage of Man fluctuated and was not significantly different between day 3 and 8.

Table 2 Effects of carbon sources and proportions in culture medium on monosaccharide mole percentage of G. lucidum EPS^a

Carbon source (g g^−1)	Time (day)	Mole percentage
		Man	Glc	Gal
a Within a column, different superscript letters (a, b, c) indicate significant differences (p < 0.05).
Glc + Gal (1 : 1)	3	2.01 ± 0.07^c	53.78 ± 5.02^a	44.21 ± 1.74^b
	6	10.3 ± 0.42^b	44.3 ± 4.17^a	45.4 ± 2.21^b
	8	12.91 ± 0.24^a	24.72 ± 3.58^b	57.92 ± 3.17^a
Glc + Gal (1 : 2)	3	4.17 ± 0.28^b	37.31 ± 1.58^a	57.35 ± 2.06^b
	6	10.08 ± 0.24^a	28.92 ± 3.69^a,b	59.08 ± 2.25^a
	8	10.47 ± 0.45^a	19.81 ± 2.72^b	67.79 ± 4.1^a
Glc + Man (1 : 1)	3	47.2 ± 1.2^a	52.8 ± 3.73^a	—
	6	52.71 ± 3.38^a	41.2 ± 3.83^a	5.67 ± 0.41^b
	8	48.35 ± 3.42^a	42.73 ± 1.95^a	7.56 ± 0.21^a
Glc + Man (1 : 2)	3	66.2 ± 4.61^a	33.8 ± 3.14^a	—
	6	52.53 ± 2.62^a	30.28 ± 0.89^a	5.73 ± 0.3^b
	8	62.18 ± 3.31^a	22.67 ± 1.26^b	12.31 ± 0.81^a

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3.2. Relationships between activities of key enzymes and mole percentage of Man and Gal in G. lucidum EPS under different carbon source conditions

Based on data for monosaccharide compositions and enzyme activities, relationships between monosaccharide mole percentages and activities of EPS synthesis related enzymes (PGI, PMI, GMP, PGM) under the four carbon source conditions were evaluated and presented as correlation coefficient (R²). Under Glc + Gal 1 : 1 and 1 : 2, activities of three of the enzymes were closely related to Man mole percentage; R² values were 0.562 for PGI, 0.807 for PMI, and 0.686 for GMP (Fig. 1). Under Glc + Man 1 : 1 and 1 : 2, only PGM activity was associated with Gal mole percentage (Fig. 2). These findings indicate that the pathway and catalyzing enzymes for Gal and Man synthesis differ. The synthesis pathway for Man in G. lucidum EPS is more complicated than the pathway for Gal, and Man mole percentage is regulated by several enzymes. No notable correlations were observed (R² < 0.5) between mole percentages of other monosaccharides (Glc, arabinose, fucose) and activities of the nine tested enzymes under any of the carbon source conditions.

Fig. 1 Relationships between activity of EPS synthesis enzymes and Man mole percentage under Glc + Gal 1 : 1 and 1 : 2. (a) PGI activity. (b) PMI activity. (c) GMP activity.

Fig. 2 Relationship between PGM activity and Gal mole percentage under Glc + Gal 1 : 1 and 1 : 2.

3.3. Effects of carbon source conditions on activities of EPS synthesis enzymes

Activities of nine G. lucidum EPS synthesis enzymes were tested under various carbon source conditions, and results for four of the enzymes were shown in Fig. 3 and 4. At the early stage (day 3) of culture, relative activities of the four enzymes were similar under Glc + Gal or Glc + Man 1 : 1 vs. 1 : 2. However, after day 5 a higher proportion of Gal in the Glc + Gal experiments was associated with increased PGI, PMI, and GMP activities. In contrast, after day 5 a higher proportion of Man in the Glc + Man experiments was associated with reduced PGM activity. These findings indicate that monosaccharide composition changes in G. lucidum EPS related to carbon source are affected by activities of related enzymes in EPS synthesis.

Fig. 3 Activities of PGI (a), PMI (b), and GMP (c) under Glc + Gal 1 : 1 (●) and Glc + Gal 1 : 2 (■).

Fig. 4 PGM activity under Glc + Man 1 : 1 (●) and Glc + Man 1 : 2 (■).

3.4. Expression levels of genes encoding key EPS synthesis enzymes

Several studies have demonstrated that monosaccharides of G. lucidum EPSs are synthesized from carbon sources in the culture medium, and that the synthesis involves several steps catalyzed by several enzymes.^15–17 To evaluate the effects of carbon sources on expression of key enzymes, we used qRT-PCR to analyze expression levels of four genes that encode those enzymes: pgi, pmi1, pmi2, and pgm. mRNA levels were normalized against rns level on day 3. Expression level of each of the four enzymes peaked on day 6 (Fig. 5). Under Glc + Gal, a higher proportion of Gal was associated with reduced levels of pmi1 and pmi2 and increased level of pgi on day 6. In comparison with the other genes, the level of pgm on day 6 was changed slightly under each carbon source condition.

Fig. 5 Transcription levels of PGI (a), PMI1 (b), PMI2 (c) and PGM (d) under four carbon source conditions. * indicate significant differences (p < 0.05).

4. Discussion

G. lucidum EPSs are bioactive compounds that have been applied for treatment of many human diseases.^4,5 Culture medium and conditions affect monosaccharide composition and molecular weight of EPSs from filamentous fungi, and thereby influence their biological activity.⁸ For industrial and medical applications, it is desirable to attain uniform chemical and biological properties of these EPSs. Among various nutrients found in culture medium, carbon source is an important factor determining monosaccharide content of EPSs. Many studies indicate that the primary monosaccharide component in EPSs does not necessarily reflect the carbon source in culture medium. In Phellinus linteus culture, the primary monosaccharide of EPSs was Glc under five different carbon sources: Glc, Gal, Man, arabinose, and starch.⁹ In Paecilomyces hepiali, a high percentage of Man in EPSs was observed under Glc carbon source, and a high percentage of Glc was observed under Man carbon source.¹⁰ In our previous study, Glc was the primary monosaccharide among three main monosaccharides (Glc, Gal, Man) in G. lucidum EPSs under Glc carbon source.¹¹ The use of sugar as carbon source was associated with an excess of the same monosaccharide in G. lucidum EPS. In the present study, we therefore examined the Gal synthesis process under Glc + Man carbon source, and the Man synthesis process under Glc + Gal carbon source, by analyzing monosaccharide composition and activities of related enzymes in EPS synthesis.

Monosaccharide analysis results showed that the major monosaccharides in G. lucidum EPSs were Glc, Gal, and Man under four mixed carbon source conditions, similar to results using Glc carbon source in our previous study.¹¹ This finding indicates a fairly uniform EPS monosaccharide composition in G. lucidum. Although carbon source had little effect on identity of monosaccharide components in EPSs, mole percentages of the major monosaccharides varied depending on carbon source conditions. The reduction in Glc mole percentage indicates that Glc is preferred over Gal or Man as carbon source for G. lucidum consuming. In experiments with Glc + Gal and Glc + Man, Gal mole percentage increased from day 3 to 8, whereas Man mole percentage fluctuated, suggesting that the metabolic pathways of Gal and Man differ. In our previous report, Glc carbon source resulted in high Glc content in G. lucidum EPSs, and there was no notable correlation of Glc mole percentage with activities of nine tested EPS synthesis enzymes.¹¹ In the present study, mole percentages of EPS monosaccharides that were the same as used carbon source sugars showed little correlation with tested enzyme activities. These findings suggest that use of sugars as carbon source results in abundance of corresponding monosaccharides in G. lucidum EPSs, obscuring the calculation of correlation coefficients between monosaccharide mole percentages and activities of EPS synthesis enzymes.

In our previous study, changes in PGM, PGI, and PMI activities were closely related to Gal and Man mole percentages in G. lucidum EPSs according to correlation coefficients calculated under various culture temperatures and initial pH values.¹¹ In contrast, PGI and PMI activities under various carbon source conditions in the present study were correlated only with Man mole percentage, and PGM activity was correlated only with Gal mole percentage. GMP activity, which had a low correlation coefficient (<0.5) with mole percentages of all monosaccharides in our previous study, had a stronger correlation with Man mole percentage in the present study. The results of the two studies, considered together, indicate that PGM, PGI, and PMI regulate mole percentages of Gal and Man in EPSs under various culture conditions and carbon sources, whereas GMP regulates mole percentage of Man only in response to carbon source. Thus, key enzymes that regulate monosaccharide synthesis pathways in G. lucidum EPSs can be divided into two groups, based on responses to culture conditions and culture medium.

Production of chemically and biologically uniform EPSs requires knowledge of EPS synthesis pathways and regulatory mechanisms. Polysaccharide synthesis pathways in bacteria have been reconstructed based on identification of intermediate compounds and activities of synthesis related enzymes.^12,13 Similar methods were used to analyze the metabolic pathway of ganoderic acids in G. lucidum.^18,19 Biosynthetic pathways have also been proposed based on the available bacterial EPS pathways and partially elucidated for some fungal EPS such as pullulan and G. lucidum EPS.^20,21 Based on these literature references and the findings from our recent study on the G. lucidum EPS,¹¹ we propose a simplified biosynthesis pathways for the three major monosaccharides (Glc, Gal, Man) in G. lucidum EPS (Fig. 6). According to this model, Glc that enters the cell is transformed to Glc-6-P, which then undergoes catalysis by PGM and PGI to form Glc-1-P and Frc-6-P, respectively. Glc-1-P is the essential precursor of UDP-Glc and UDP-Gal, the major monosaccharides of G. lucidum EPSs. Frc-6-P participates in energy metabolism via the tricarboxylic acid cycle, and is the precursor of UDP-Man as catalyzed by PMI, GMP, and other enzymes.

Fig. 6 Proposed biosynthesis pathways of sugar nucleotides for G. lucidum EPSs. -P, -phosphate. Frc, fructose. TCA, tricarboxylic acid cycle.

The contents of Gal and Man in G. lucidum EPSs were regulated by both PGM and PGI which were affected by culture conditions.¹¹ As shown Fig. 6, PGM and PGI activities determine metabolic flux from Glc-6-P to two different and competing pathways, i.e., Gal and Man synthesis pathways. Thus, differing culture conditions and resulting changes in metabolic flux jointly determine monosaccharide composition through key enzyme activities. In microorganisms, Gal carbon source is metabolized via the Leloir pathway and participates in EPS synthesis through transformation to Glc-1-P, as catalyzed by galactokinase, uridylyltransferase, and UDP-Gal 4-epimerase.²² In G. lucidum mycelia, the presence of Gal in a carbon source mixture may increase the level of intermediate Glc-1-P, and metabolic flux of Glc-6-P to UDP-Gal. Resulting feedback inhibition may increase flux from Glc-6-P to GDP-Man, thereby enhancing activities of PGI, PMI, and GMP. In microorganisms, Man-6-P is directly formed from Man through catalysis by several weakly specific enzymes, such as hexokinases (HXK1, HXK2) and glucokinase.²³ Presence of Man in a carbon source mixture increases metabolic flux to GDP-Man. However, the precursor of the GDP-Man synthesis pathway is a downstream intermediate of Frc-6-P, which also participates in the tricarboxylic acid cycle. Metabolic flux stress has little effect on the competing pathway (UDP-Gal synthesis pathway); this may explain why higher Man ratio in the carbon source mixture results in reduced PGM activity.

In the present study, activities and encoding gene expression levels for four key EPS synthesis enzymes were compared under four carbon source conditions. In contrast to the constantly increasing enzyme activities, expression levels of the genes peaked on day 6 and then declined. Gene expression in cells is determined by firstly transcription and then translation. The apparent discrepancy between encoding gene transcription and activity levels of the four key enzymes may therefore arise at the translational level. Environmental factors may also affect monosaccharide mole percentages of G. lucidum EPSs by operating at the translational level of key enzymes.

5. Conclusion

In submerged fermentation of G. lucidum, major monosaccharides of EPSs are determined by the carbon source (or sources) in the culture medium. In the experiments with various carbon source conditions, correlation analysis showed that mole percentage of Gal in EPSs was determined by PGM, and mole percentage of Man was determined by PGI, PMI, and GMP; these enzymes are involved in the Gal and Man synthesis pathways, respectively. Observed differences in activities of key synthesis enzymes and expression levels of the encoding genes under differing carbon source conditions suggest that monosaccharide composition of G. lucidum EPSs may be controlled at the translational level. Our findings are helpful for understanding Gal and Man synthesis pathways and key enzymes in G. lucidum EPS synthesis, and for attaining uniform monosaccharide composition and biological activity of G. lucidum EPSs in pharmaceutical, medical, and industrial applications.

Abbreviations

EPS	Exopolysaccharide
Gal	Galactose
Glc	Glucose
GMP	GDP-D-Man pyrophosphorylase
Man	Mannose
PGI	Phosphoglucose isomerase
PGM	α-Phosphoglucomutase
PMI	Phosphomannose isomerase

Acknowledgements

Funding for this study was provided by the National Natural Science Foundation of China (31271918 and 21206177), National High Technology Research and Development Program of China 863 Program (2012AA021505), Fundamental Research Funds for the Central Universities (JUSRP51411B), and Open Project Program of Key Laboratory of Carbohydrate Chemistry and Biotechnology (KLCCB-KF201205). The authors are grateful to Dr S. Anderson for English editing of the manuscript.

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Footnote

† These two authors made equal contributions to the study.

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