Nitrogen Source Affects Glycolipid Production and Lipid Accumulation in the Phytopathogen Fungus Ustilago Maydis

Advances in Microbiology, 2014, 4, 934-944 Published Online October 2014 in SciRes. http://www.scirp.org/journal/aim http://dx.doi.org/10.4236/aim.2014.413104 Nitrogen Source Affects Glycolipid Production and Lipid Accumulation in the Phytopathogen Fungus Ustilago maydis Ariana Zavala-Moreno1, Roberto Arreguin-Espinosa2, Juan Pablo Pardo3, Lucero Romero-Aguilar1, Guadalupe Guerra-Sánchez1* 1Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, IPN, México D.F., Mexico 2Departamento de Química de Biomacromoléculas, Instituto de Química, UNAM, Ciudad Universitaria, México D.F., Mexico 3Departamento de Bioquímica, Facultad de Medicina, UNAM, Ciudad Universitaria, México D.F., Mexico Email: *[email protected] Received 3 August 2014; revised 2 September 2014; accepted 5 October 2014 Copyright © 2014 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ Abstract When cultured in medium limited of nitrogen sources, the phytopathogen Ustilago maydis produces two amphipathic glycolipids: Ustilagic acid (UA) and Mannosylerythritol lipid (MEL), which in addition to the hydrophilic moiety, contain di- or tri-hydroxylated C16 fatty acids (UA), or C8 and C16 saturated fatty acids (MEL). We compared the growth and morphology of cells in YPD and in minimum media containing glucose and nitrogen sources such as nitrate or urea and those deprived of nitrogen. Nitrogen-starved cells showed a dramatic accumulation of internal lipids iden- tified as lipid droplets when stained with the hydrophobic probe BODIPY; these lipid droplets were enriched in unsaturated fatty acids. Fatty acids in YPD or medium containing nitrate as nitrogen source showed a combination of saturated/unsaturated lipids, but when urea was the nitrogen source, cells only contained saturated fatty acids. The glycolipid profiles produced in the presence or absence of nitrogen showed preferences towards the production of one kind of glycolipid: cells in media containing nitrate or urea produced different proportions of UA/MEL, but under nitrogen starvation cells contained only UA. The emulsification capacity of the glycolipids produced in media with or without nitrogen was similar (72% - 76%). HPLC of the glycolipids allowed the separation of fractions with different emulsifying characteristics. Our results indicate that U. maydis accumulates lipid droplets when deprived of nitrogen source and confirm that UA is not under nitrogen control, but rather that MEL and lipid droplets are produced and oppositely regu- lated by nitrogen. *Corresponding author. How to cite this paper: Zavala-Moreno, A., Arreguin-Espinosa, R., Pardo, J.P., Romero-Aguilar, L. and Guerra-Sánchez, G. (2014) Nitrogen Source Affects Glycolipid Production and Lipid Accumulation in the Phytopathogen Fungus Ustilago maydis. Advances in Microbiology, 4, 934-944. http://dx.doi.org/10.4236/aim.2014.413104 A. Zavala-Moreno et al. Keywords Nitrogen Starvation, Yeast Glycolipids, Lipid Droplets, Lipid Accumulation 1. Introduction The yeast of Ustilago maydis produces extracellular glycolipids with surfactant activity that enhance the availability of hydrophobic nutrients during its interaction with the host [1] [2]. These biosurfactants are derivatives of two classes of amphiphatic glycolipids: Ustilagic acid (UA) and Mannosylerythritol lipids (MEL). They are secreted into the extracellular media at the stationary phase when nitrogen becomes an environmental limitation [3]. Their surfactant properties make this type of glycolipids very promising for commercial production in cos- metic and pharmaceutical industry [4]-[6]. In recent years, several reports described enhanced yields of these biosurfactants when U. maydis cells are grown in yeast hydrolysates, urea and nitrate or ammonium salts, as nitrogen sources, and using also multiple carbon sources such as glucose, sucrose, lipids and glycerol [7]-[9]. Dif- ferent strains have been used for the production of glycolipids and the yield of the two glycolipids may be af- fected by the availability of nitrogen source [10]. The biosynthesis of UA and MEL depends on specific gene clusters [1] [11] [12]. For UA, cyp1 and cyp2 genes are required for terminal and subterminal hydroxylation of hexadecanoic acid [12]. Mannosylerythritol lipids (MEL) are secreted in at least four different varieties (ABCD), depending on the number of acetyl groups attached to the disaccharide [3]-[11] and a glycosyltransferase is es- sential for the biosynthesis of MEL; expression of this enzyme is strongly induced by limitations on nitrogen availability [11]. To produce these glycolipids, U. maydis requires raw materials to synthesize the lipid and car- bohydrate moieties. Different carbon sources have been used in an effort to attain high yields of these glycolipids [3]-[9], but less attention has been placed on identifying the effect of nitrogen sources on the production of glycolipids. When cells are limited in terms of their nitrogen sources, neutral lipids are accumulated as discrete deposits, named lipid droplets (LD) [13] [14]. Moreover, when cells are switched from a rich media to a minimal medium lacking of nitrogen source, autophagy is induced to cope with the starvation condition [15]. In this work, we studied the growth and morphology of U. maydis yeasts cultured in YPD and minimal media (MM) containing glucose as carbon source without a nitrogen source (MM−N), or glucose under nitrogen limiting conditions using nitrate (MM+N) or Urea (MM+U) as nitrogen sources. We also reported the presence of lipid droplets, accumulation of unsaturated fatty acids and production of the biosurfactant Ustilagic acid (UA) in cells deprived of nitrogen. Glycolipids profile was altered by the presence of nitrogen in the culture media, with the production of mixtures of UA and MEL, and changes in the saturated/unsaturated fatty acids ratio. The emulsifying capacity of glycolipids synthesized by cells grown either under nitrogen limiting conditions (nitrate or urea) or deprived of nitrogen was also evaluated. This study confirmed that in contrast to MEL, UA is not under nitrogen control, and that U. maydis is a potential microorganism for the production of lipids for biofuels and biosurfactants in nitrogen deprived conditions. 2. Materials and Methods 2.1. Chemicals All reagents were reactive or HPLC grade from JT Baker or Sigma Chem Co. Surfactin from Bacillus subtilis was purchased from Sigma. BODIPY (4, 4-difluoro-3a, 4a,-diaza-s-indacene) 483/503 was from Invitrogen and prepared as a 10 mM stock solution in DMSO and kept at −70˚C. For assay of total lipids, a clinical kit was obtained from Spinreact. TLC plates of Silica-Fluorescent gel 60 were from Merck. 2.2. Strain and Culture Condition The Ustilago maydis ATCC 201384 FB2 strain was obtained from American Type Culture Collection and kept in 25% glycerol at −70˚C. Cells were streaked in YPD plates (10 g∙L−1 glucose, 1.5 g∙L−1 ammonium nitrate, 2.5 g∙L−1 peptone, 10 g∙L−1 yeast extract and 62.5 ml∙L−1 of salt solution adjusted to pH 7.0 and agarose 2%), and incubated at 28˚C for 24 h. They were subsequently transferred to 100 ml YPD media and shaken at 125 rpm at 29˚C by 24 h. This pre-culture was used as inoculum for all further experiments. Minimum media with nitrate 935 A. Zavala-Moreno et al. (MM+N) contained 0.3% potassium nitrate, 1% glucose and 62.5 ml∙L−1 of salts solution [16], adjusted to pH 7.0 with 1 N NaOH. The minimum media without a nitrogen source (MM−N) contained 1% glucose and salts solution at pH 7.0. Minimum media with urea (MM+U) contained 1% glucose, 0.06% urea and same salts solution. The growth curve was determined by inoculating 50 OD600 in 1 L and incubated for 7 days in a rotary shaker (125 rpm) at 29˚C. Aliquots of 1 ml were taken to measure the growth of cells by optical density at 600 nm and the morphology of cells was monitored by light microscopy (100×) in 6 - 12 h time intervals during the culture period. Statistical analysis was performed using Excel 2007. Data was analyzed using overall one-way analysis of variance (ANOVA) and the differences observed were significant at P ≤ 0.05. 2.3. Fluorescence Microscopy of Lipid Droplets in U. maydis Cells For the determination of lipid droplets by fluorescense microscopy, aliquots were withdrawn during growth and immediately treated as described in [17]. Briefly, aliquots of 5 ml of cellular suspension (10 OD600) were fixed with 24% formaldehyde, centrifugated at 2500 g and washed with H2O milliQ (2×). Cells were re-suspended at 10U OD and 10 µl were mixed with 200 µl of 50 mM KI and 5 µl of BODIPY. Cells were mounted onto a po- lylysine treated glass slide. Microscopy was performed on Olympus 1× 81 at 100× oil immersion objective. The captured images were analyzed using Image software. 2.4. Measurement of Total Lipids and Identification by GC/MS Cultures were grown in the media of YPD, MM+N, MM−N and MM+U and cells harvested by centrifugation at 2500 g for 10 min. 40 mg of cells from each media (wet weight) were re-suspended in 300 µl of 50 mM HEPES buffer pH 7.0 and equal mg of glass beads (0.5 mm, Biospec) were added and lyzed by vortexing for 10 cycles of 1 min at 4˚C. Lysed cells were separated by centrifugation at 2500 g for 15 min and the glass beads were washed with 100 μl of the same HEPES buffer and spun. The total lysate was centrifuged at the same condition to get the supernatant. Lipids were extracted using the final supernatant and mixed with chloroform/methanol/ water (65:25:4) solution. After shaking for 5 minutes, the organic phase was separated by centrifugation and the solvent was evaporated under vacuum.

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