Production and identification of mannosylerythritol lipid-A homologs from the ustilaginomycetous yeast Pseudozyma aphidis ZJUDM34

Carbohydrate Research 392 (2014) 1–6

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Production and identification of mannosylerythritol lipid-A homologs from the ustilaginomycetous yeast Pseudozyma aphidis ZJUDM34 Lin-Lin Fan a, Ya-Chen Dong a, Yi-Fei Fan a,b, Jun Zhang a, Qi-He Chen a,b,⇑ a b

School of Biosystems Engineering and Food Science, Zhejiang University, Yuhangtang Rd. 866, Hangzhou 310058, PR China Fuli Institute of Food Science, Zhejiang University, Hangzhou 310058, PR China

a r t i c l e

i n f o

Article history: Received 7 March 2014 Received in revised form 17 April 2014 Accepted 18 April 2014 Available online 29 April 2014 Keywords: Mannosylerythritol lipid-A Pseudozyma aphidis Glycolipid Homologs Biosurfactant Bioemulsifier

a b s t r a c t Mannosylerythritol lipids (MELs) are mainly produced by strains of the genus Pseudozyma and by Ustilago maydis. These glycolipid biosurfactants exhibit not only excellent surface-active properties but also versatile bioactivities. Mannosylerythritol lipid-A (MEL-A) is worth investigating due to its self-assembling property. In this work, crude MELs were produced by resting Pseudozyma aphidis ZJUDM34 cells using different culture media. MEL-A fractions were isolated and identified using high-performance liquid chromatography combined with mass spectrometry (HPLC–MS) and gas chromatography combined with mass spectrometry (GC–MS). The results showed that MEL-A homologs had long unsaturated fatty acid chains, and the chain lengths range from C8 to C20. Nuclear magnetic resonance (NMR) was employed to confirm the chemical structures of the MEL-A homologs. Fermentation medium without NaNO3 and medium with manganese ions enhanced MEL-A production by Pseudozyma aphidis ZJUDM34. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Mannosylerythritol lipids (MELs) are oily compounds that are secreted in abundance by strains of the genus Pseudozyma and by Ustilago maydis. Pseudozyma aphidis has been reported to produce large amounts of MELs (the maximum 165 g L1) in fed-batch bioreactor cultivation.1 This nonionic biosurfactant is composed of the polar 4-O-b-D-mannopyranosyl-D-erythritol and two nonpolar fatty acid acyl chains.2,3 They are called MEL-A (diacetylated at O-4 and O-6), MEL-B (monoacetylated at O-6), MEL-C (monoacetylated at O-4), and MEL-D (deacetylated) depending on the number of acetyl groups and their locations on the mannose (Fig. 1). MEL-A has various chemical structures due to variations in fatty acid chain length and degree of unsaturation.4 For example, Pseudozyma rugulosa NBRC 10877 produced MEL-A with different types of fatty acids, such as C8:0 (28.1%), C10:0 (21.7%), and C10:1 (22.9%), as the hydrophobic moiety.5 MEL-A produced by Ustilago maydis DSM 4500 contained fatty acids with C14:1 (43.0%), C6:0 (20.0%), and C16:1 (12.0%).6 Thus, the properties of MEL-A homologs vary with the types of fatty acids they contain. As a biosurfactant, MEL-A shows excellent interfacial properties and pharmacological activities. It can self-assemble into distinctive ⇑ Corresponding author. Tel.: +86 571 86984316. E-mail address: [email protected] (Q.-H. Chen). http://dx.doi.org/10.1016/j.carres.2014.04.013 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.

lyotropic liquid crystals including sponge, bicontinuous cubic, and lamella phases. In this property, MEL-A is superior to MEL-B/C. Interestingly, MEL-A is the first natural compound to display the formation of the sponge phase by itself.7,8 Although MEL-A has poor solubility in water, it can form a stable W/O microemulsion in the ternary MEL-A/water/n-decane phase system without other cosurfactants.9 MEL-A was reported to dramatically increase gene transfection via membrane fusion and had cell differentiation activity against human leukemia cells, mouse melanoma, PC12 cells, or other damaged cells.10 Accordingly, it has been used as an additive in cosmetics. Pseudozyma aphidis is well known as an industrial producer of MEL biosurfactants, and this strain is potentially advantageous for use in MEL-A synthesis. In this work, MELs were produced by resting Pseudozyma aphidis ZJUDM34 cells in different cultivation media. MEL-A homologs were characterized, and their chemical structures were elucidated by LC–MS, GC–MS, and 1D NMR. 2. Materials and methods 2.1. Microorganism Pseudozyma aphidis was acquired from Deutsche Stammsammlung für Mikroorganismen und Zellkulturen, Braunschweig, Germany. The Pseudozyma aphidis ZJUDM34 mutant was evolved

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CH3 H 3C

1 2

n

H

O

OR1 n

H O

O

3'

CH 2

O

5'

R 2O

OH 4

O 2'

methanol:water (75:15:2, v/v). The isolated compounds on the TLC plates were located by charring at 105 °C for 5 min after spraying the plates with an orcinol reagent with sulfuric acid.

OH 3

6' O

4'

CH2OH

1'

Figure 1. Structure of MELs. MEL-A: R1 = R2 = Ac; MEL-B: R1 = Ac, R2 = H; MEL-C: R1 = H, R2 = Ac; MEL-D: R1 = R2 = H, n = 6–16.

from wild-type yeast using UV mutagenesis and low-energy N+ implantation methods. It exhibited enhanced ability to produce MEL-A. This mutant was stored in our lab. Stock cultures were grown at 28 °C for 4 days on agar medium plates containing 3.0 g yeast extract, 3.0 g malt extract, 5.0 g peptone, 10.0 g glucose, 15.0 g agar, and 1.0 L distilled water. Stock cultures were stored at 4 °C and renewed every 4 weeks. 2.2. Cultivation conditions and biosynthesis of MELs Seed cultures were prepared by inoculating cells previously grown on an agar plate in 500-mL flasks. The preculture medium contained 40.0 g/L glucose, 3.0 g/L NaNO3, 0.3 g/L MgSO47H2O, 0.3 g/L KH2PO4, and 1.0 g/L yeast extract (pH 6.0), and the cultures were incubated at 28 °C on a rotary shaker (220 rpm) for 3 days. Then, 1% (g/v) resting cells were washed twice with 0.9% NaCl solution and then transferred to basal liquid culture medium followed by incubation on a rotary shaker (220 rpm) at 28 °C for 10 days. In order to identify the optimal cultivation condition, different basal cultivation media were designed in this study. Experiments were carried out in 500 mL shake flasks containing 100.0 mL cultivation medium. The compositions of the liquid media were as follows: YNB-S culture medium contained 1.7 g/L yeast nitrogen base without amino acids and 80.0 mL/L soybean oil, pH 6.0; YNB-T culture medium consisted of 1.7 g/L yeast nitrogen base without amino acids and 80.0 mL/L blend oil, pH 6.0; BM-S culture medium contained 10.0 g/L yeast extract, 2.0 g/L NaNO3, 0.2 g/L MgSO47H2O, 0.2 g/L KH2PO4, and 80.0 mL/L soybean oil, pH 6.0; BM-T culture medium consisted of 10.0 g/L yeast extract, 2.0 g/L NaNO3, 0.2 g/L MgSO47H2O, 0.2 g/L KH2PO4, and 80.0 mL/L blend oil, pH 6.0; PBS culture medium consisted of 10.0 g/L yeast extract, 4.0 g/L peptone, 1.0 g/L MgSO47H2O, 0.5 g/L KH2PO4, 2.0 g/L MnSO4, 0.4 g/L CuSO4, and 80.0 mL/L soybean oil, pH 6.0.

2.4. Structural characterization of MELs 2.4.1. Mass analysis by LC–MS The purified compounds were used for LC–MS analysis. Agilent1200 HPLC system (Agilent Technology, USA) was connected to a MS spectrometer (LCD Deca xp max, Thermo Electron Corporation). A 5 lm (250 mm  4.6 mm) Agilent ZORBAX SB-C18 column was used. The mobile phase consisted of solvent A (isopropanol) and solvent B (acetonitrile). The elution was conducted at a flow rate of 0.2 mL/min in a two-step ascending linear gradient as follows: solvent A from 10% to 30% within 10 min and then to 100% within 55 min. The ionization parameters were adapted to the flow rate and the mass range (200–1500). A drying temperature of 325 °C was applied together with a drying gas (N2) at a flow of 10 mL/ min, a capillary voltage of 2500 V, a corona voltage of 4000 V, and a nebulizer pressure of 35 psi. The injection volume was 5 lL.11 2.4.2. Fatty acid analysis by GC–MS The MEL components were methanolyzed by an alkaline method to synthesize the methyl ester derivatives. In detail, 1.0 mL of the purified MEL fraction was mixed with 10.0 mL of NaOH (2 N in methanol). After stirring at room temperature for 1 h, the mixture was extracted 3 times with hexane, and 10.0 lL of the extract was injected into the GC–MS using an Agilent HP5MS column (30 m  0.25 mm  0.25 lm). The oven temperature was maintained at 100 °C for 2 min, then increased from 100 to 180 °C at 15 °C/min and maintained at 180 °C for 1 min. Next, the temperature was increased from 180 to 300 °C at 5 °C/min and held for 10 min. A scan range of 40–600 amu and a source temperature of 250 °C were used. 2.4.3. MEL analysis by NMR The refined glycolipid (10.0 mg) was dissolved in 0.5 mL CDCl3 (99.9%). Both 1H NMR and 13C NMR spectra were recorded at 25 °C by a Bruker AVIII 500M instrument with TMS as an internal standard.

120

2.3. Isolation and purification of MELs

80

Yield(g/L)

After submerged fermentation, the culture broth was extracted with an equal volume of ethyl acetate. The organic layer was separated and evaporated. The amount of crude MELs was estimated by precisely weighing the ethyl acetate fraction after completely removing the solvent via evaporation. All measurements reported herein are calculated from three independent experiments. The sticky MELs were then washed with n-hexane and methanol twice to remove the remaining oil and fatty acids. After the removal of methanol and water by vacuum evaporation, 2.0 g of the mixture was dissolved in 5.0 mL of chloroform and fractionated on a glass column (3 cm  40 cm) loaded with 50.0 g silica gel. Gradient elution with a chloroform/methanol (from 10:0 to 0:10, v/v) mixture was performed. After the above refining steps, the MEL extracts were analyzed by thin-layer chromatography (TLC) on silica plates (Silica gel 60F) in two steps. The first solvent system was petroleum ether, and the second consisted of chloroform:

100

60

40

20

0 BM-S

BM-T

YNB-S

YNB-T

PB-S

Figure 2. Comparison of crude MELs from different submerged culture media. Every culture media was inoculated with 1.0% (g/v) wet cells that were washed twice with 0.9% NaCl solution after the seed culture and then incubated on a rotary shaker at 220 rpm at 28 °C for 10 days.

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928.95

931.05

(a)

90

(c)

(b)

80 Relative Abundance

901.31

961.52

100

894.09

972.72

70 60

(d) 970.64

896.22

50 40 30

878.08

20

936.79

10

1.01

0 200

987.23

880.51 986.61

853.53 1000

800

m/z

984.40 1012.54

770.11 800

1000

.69 876.19

1000

800

m/z

m/z

942.50 955.69 1000

m/z 831.41

898.98

897.15

(e)

849.30

(f)

(g)

(h)

901.11

873.06 856.13 800

940.64 982.33 1000

m/z

880.16

938.59 980.55

800

1000

831.77 800

m/z

927.81

948.33 974.15

816.43

1000

800

900

1000

m/z

m/z

Figure 3. Mass spectra of MEL-A homologs (a–h) isolated from the PB-S liquid cultivation medium.

Table 1 Detected mass of MEL-A

Table 2 Types of partial fatty acids detected by GC–MS with corresponding retention times

[M+H]+

Molecular mass

Possible fatty acids chain combinations

Retention time (min)

Fatty acid profile

Relative content (%)

961.5 931.1 928.9 901.3 898.9 897.1 894.8 849.3 831.4 737.1

960.5 930.1 927.9 900.3 897.9 896.1 893.8 848.3 830.4 736.1

20:1–20 18:1–20:1 18:2–20:1/18:1–20:2 18:2–18:1/18:0–18:3/16:1–10:2 18:2–18:2/18:1–18:3 18:3–18:2 18:3–18:3 16:3–16:2 12:0–18:0/14:0–16:0 14:3–10:2/10:3–14:2/10:3–12:2

4.32 5.19 6.13 6.37 8.48 10.75 10.92 13.42 13.93 17.20 18.21 20.05

C8:0 C12:1 C12:0 C12:2 C10:2 C14:0 C14:2 C16:1 C16:0 C18:0 C18:1 C20:0

10.7 6.4 5.4 1.2 4.2 0.1 0.1 9.2 8.6 13.1 23.6 12.7

Fatty acid combinations of the MEL-A homologs were deduced.

2.5. Statistical analysis The error bars in the figures represent the standard error of the mean (SEM). Analysis of variance was performed using ANOVA. 3. Results and discussion 3.1. MELs produced from different submerged culture media Five types of media were optimized for MEL production. After submerged fermentation, the culture broths were extracted, and

the amount of crude MELs was calculated. Figure 2 shows that more MELs were produced in the PB-S fermentation medium than other media. The presence of mineral salts and the fermentation conditions contributed to these differences. The type of nitrogen source also significantly affected MEL production. The PB-S medium contained peptone as the main nitrogen source rather than NaNO3. Other studies have reported that the highest MEL-B and MEL-C yields were achieved with NaNO3 as the nitrogen source, but the MEL-A yield was relatively low.12 Thus, the type of nitrogen source or the ratio of carbon to nitrogen may affect MEL formation.

Figure 4. Partial

13

5.0

4.0 3.661

3.0

2.0

1.0 0.081 0.011

110

0.777 0.763

99.782

20

0.909 0.902 0.887

120

1.277 1.268

1.332 1.321 1.309

30

1.651 1.638 1.625

130

1.877 1.693

140 130.796 129.155 128.987

133.047 131.309 131.106

40

2.117 2.067 2.014 2.003

150

2.325 2.312 2.298 2.147

50

2.418 2.405 2.393

160

3.591 3.578 3.563 2.772

170

3.759 3.750

168.827 168.668

173.937 171.495

60

3.786 3.780

180

3.968 3.963 3.908 3.899 3.806

175.810 174.982 174.397 174.354

70 ppm (t1)

4.463 4.456 4.446

4.718

4.933

5.320

5.507 5.502 5.400

71.751

73.103

19.661 15.187 15.144

21.038

23.762 23.652

25.954 25.775

26.714

28.287

32.608 30.849 30.784 30.708 30.606 30.427 30.400 30.259 30.204 30.172 30.119

34.869 33.637 32.986

35.278 35.173 35.114

59.562

63.192

69.492 66.134

69.982

71.302

4 L.-L. Fan et al. / Carbohydrate Research 392 (2014) 1–6

10

(a)

ppm (t1) 100

(b)

ppm (t1)

0.0

(c)

C NMR spectrum of MELs: signals at 10.0–75.0 ppm (a) and 100.0–180.0 ppm (b); partial 1H NMR spectrum of MELs: signals at 0.0–5.5 ppm (c).

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3.2. Isolation and TLC verification of MELs Crude MELs were spotted for TLC. Four spots in crude MELs with Rf values of 0.7, 0.5, 0.5, and 0.3 were observed (data not shown). This result was consistent with the data reported by Kitamoto et al.13 The four spots are MEL-A, MEL-B, MEL-C, and MEL-D, respectively. It should be noted that another unknown spot was present at the top of silica plate, most likely caused by free fatty acids. To obtain purified MEL fractions, the eluent was collected in 10.0 mL fractions from a silica column and spotted for TLC. The fraction with an Rf value 0.7 was identified as MEL-A. All constituents with similar Rf values were collected together, representing approximately 74.6% of the total MELs in volume. This result confirms that of a previous study that showed that MEL-A was the main metabolic product secreted by Pseudozyma aphidis.14 The refined fractions were used for further examination. 3.3. Identification of MELs To identify the types of MEL-A, both LC–MS and GC–MS were employed. Subsequently, the structure was determined by NMR. 3.3.1. HPLC–MS experiments and analysis The refined MEL-A fractions were analyzed by HPLC–MS. The positive ion peaks and predictable MEL-A types in the corresponding retention time are summarized. As shown in Figure 3, the ion detected for MEL-A was m/z [M+H]+ with a mass-to-charge ratio of 831, 849, 895, 897, 899, 901, 929, 931, and 961. The possible fatty acids ranged from C10 to C20 with one, two or three unsaturated bonds (shown in Table 1). Some peaks with higher masses were observed. Onghena et al.11 found that Pseudozyma aphidis could produce MEL-A with m/z 576–760, and the fatty acid chains had one or two unsaturated bonds. The mass-to-charge ratio differences are the result of the use of the PB-S fermentation medium in our work. In contrast to other cultivation media, the PB-S medium contained mineral salts such as manganese, which played a critical role in keeping enzymes and proteins active.15,16 The metal ion could affect the synthetic pathway of MEL formation. In addition, different homologs of MEL-A had 14 (ACH2A) units or a double bond unit difference. Here, the fatty acid chain length and degree of unsaturation are the crucial factors for MEL-A structural diversity. 3.3.2. GC–MS experiments and analysis Next, we analyzed the fatty acids in order to determine the exact form of MEL-A. MEL-A was methanolyzed with strong alkali-methanol solvent. n-Hexane was added to the mixture to extract fatty acid methyl esters, 5 lL of which was used for GC. Several components of long-chain fatty acids (C8–20) appeared at the appropriate retention times. The results of GC–MS analysis of fatty acid methyl esters presented in Table 2 further verified the GC results. Moreover, we can see from the table that no structures with three double bonds appeared. These substances may not be detected by GC–MS at low levels or the structure may have changed. In contrast to MEL compositions described in the literature,12 the content of C14 fatty acids with one or two double bonds was high in this study. In addition, the soybean oil used in this investigation was a mixture of multiple components, containing palmitic acid, stearic acid, arachidic acid, oleic acid, linoleic acid, linolenic acid, etc., most of which are characterized by carbon lengths of C16–C20. It is interesting to note that all fatty acids contained in the glycolipids were degraded by C2 units to become compounds with chain lengths of C8, C10, C12, C14, C16, and C18. The partial b-oxidation of fatty acids was probably involved in the fermentation process. This b-oxidation process was first described in Candida antarctica as a chain-shorting pathway.17

Table 3 H NMR and

1

13

C NMR spectral data for MELs

Structure 0

C-1 C-20 C-30 C-40 C-50 C-60 C-1 C-2 C-3 C-4 ACO-R2/R3/R4/R6 AOCOCH2-R4/R6 A(CH2)nA ACH@CHA ACH@CHACH2A ACH3(ACOCH3) ACOCH2CH2A ACO

13

C NMR d (ppm)

99.7d 69.4d 71.3d 66.1d 71.7d 62.8t 63.2t 71.8d 69.9d 73.1t 168.8–171.5m 34.8–35.2m 23.6–30.8m 129.0–131.3m 26.7t 15.1–15.2 (21.0) 23.7t 173.9–175.8m

Structure 0

H-1 H-20 H-30 H-40 H-50 H-60 H-1 H-2 H-3 H-4 A(CH2)nA ACH2CH@CHA CH3COO-R4/R6 AOCOCH2-R2/R3 ACH@CHA ACH3

The chemical shifts of MELs in 1H NMR and

13

1

H NMR d (ppm)

4.7d 5.5dd 4.9dd 4.4–4.6m 3.5–3.6m 4.3m 3.6–3.7m 3.7m 3.6m 3.6m 1.3–1.5m 1.9–2.1m 2.0–2.1b 1.7–1.9b 5.9–6.0b 0.8b

C NMR spectrum are summarized.

For other strains, the biosynthetic pathway has not been clearly demonstrated. The synthetic pathway of MELs in Pseudozyma aphidis ZJUDM34 may be similar to that of Candida antarctica. More research needs to be done to verify this metabolic process. 3.3.3. NMR analysis The components of MEL-A were analyzed in detail by 1H NMR and 13C NMR (shown in Fig. 4 and Table 3). The crucial groups that defined MEL types of were signals at 13C NMR d 168.8–171.5 (–CO– ) and d 21.0 (–COCH3). Two acetyl signals were confirmed in this study. Other 13C NMR signals were d 99.7d (C-10 ), d 69.4d (C-20 ), d71.3d (C-30 ), d 66.1d (C-40 ), d 71.7d (C-50 ), d 62.8t (C-60 ), d 63.2t (C-1), d 71.8d (C-2), d 69.9d (C-3), d 73.1t (C-4), d 23.6-30.8 (A(CH2)nA), d 129.0–131.3 (ACH@CHA), and d 15.1–15.2 (ACH3). Resonances like 1H NMR d 3.5–3.7 m were assigned to H1-4 of erythritol. d 4.7d, d 5.5dd, d 4.9dd, d 4.4–4.6m, d 3.5–3.6m, and d 4.3m were referred to H10 -60 of mannose, respectively. d 1.3–1.5m (A(CH2)nA), d 5.9–6.0b (ACH@CHA), d 1.7–1.9b (AOCOCH2), and d 0.8b (ACH3) were also detected by 1H NMR. Based on the data above, it can be stated that the MEL identified in this research was MEL-A. The chemical shifts of other signals were slightly different from those ascribed to MEL-A in previous studies.6,12 The types of fatty acids contribute to the differences in various MEL-A homologs. Considering our previous results, MEL-A possessed good stability during exposure to high and low temperatures, high salinity and a wide range of pH values at room temperature. MEL-A is particularly useful for microemulsion formation without any cosurfactants. It has great potential application as a bioemulsifier.18 Thus, new MEL-A homologs need to be given more attention in future studies. 4. Conclusion In conclusion, resting Pseudozyma aphidis ZJUDM34 cells can secrete four types of glycolipid biosurfactants from soybean oil, and MEL-A is the main product. When cultivated in a medium containing peptone, manganese and copper salts, the strain Pseudozyma aphidis ZJUDM34 exhibited better production of MEL-A homologs. However, the mechanism of MEL-A synthesis with long polyunsaturated fatty acid chains is still unclear. Therefore, further investigation of the effects of mineral salts, particularly manganese and copper salts, on the key metabolic enzymes and lipid metabolism involved in MEL-A biosynthesis is required.

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Acknowledgments This work was financially supported by the National Natural Science Foundation of China (31171734), Zhejiang Provincial Natural Science Fund of China under Grant No. LR13C200002, and Foundation of Fuli Institute of Food Science, Zhejiang University (No. KY201302). References 1. Rau, U.; Nguyen, L. A.; Roeper, H.; Koch, H.; Lang, S. Appl. Microbiol. Biotechnol. 2005, 68, 607–613. 2. Kitamoto, D. Agric. Biol. Chem. Tokyo 1990, 54, 31–36. 3. Kim, H. S.; Yoon, B. D.; Choung, D. H.; Oh, H. M.; Katsuragi, T.; Tani, Y. Appl. Microbiol. Biotechnol. 1999, 52, 713–721. 4. Arutchelvi, J. I.; Bhaduri, S.; Uppara, P. V.; Doble, M. J. Ind. Microbiol. Biotechnol. 2008, 35, 1559–1570. 5. Morita, T.; Konishi, M.; Fukuoka, T.; Imura, T.; Kitamoto, D. Appl. Microbiol. Biotechnol. 2006, 73, 305–313. 6. Spoeckner, S.; Wray, V.; Nimtz, M.; Lang, S. Appl. Microbiol. Biotechnol. 1999, 51, 33–39.

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