World J Microbiol Biotechnol (2009) 25:921–925 DOI 10.



A quick isolation method for mutants with high lipid yield in oleaginous yeast
Jufang Wang Æ Renmin Li Æ Dong Lu Æ Shuang Ma Æ Yaping Yan Æ Wenjian Li

Received: 12 September 2008 / Accepted: 7 January 2009 / Published online: 24 January 2009 Ó Springer Science+Business Media B.V. 2009

Abstract A novel method has been developed to easily isolate the mutants with high lipid yield after irradiating oleaginous yeast cells with carbon ions of energy of 80 MeV/u. Pre-selection of the mutants after ion irradiation was performed with culture medium in which the concentration of cerulenin, a potent inhibitor of fatty acid synthetase, was at 8.96 lmol/l. Afterwards, lipid concentration in the fermentation broth of the pre-selected colonies was estimated by the sulfo-phospho-vanillin reaction instead of the conventional methanol–chloroform extraction. Two mutants with high lipid yield have been successfully selected out by the combined method. This easy and simple method is much less time-consuming but very efficient in the mutant isolation, and it has demonstrated great potential on mutation breeding in oleaginous microorganism. Keywords Oleaginous yeast Á Mutant Á Cerulenin Á Sulfo-phospho-vanillin reaction

Introduction The commercial application of microbial oils, i.e. single cell oils (SCO), has become commonplace worldwide and the demand is continuously increasing (Spolaore et al. 2006). It has long been a subject of both research and
This work was supported by the ‘‘Western Light’’ Program of Talent Cultivation of Chinese Academy of Sciences (O606180XBO). J. Wang (&) Á R. Li Á D. Lu Á S. Ma Á Y. Yan Á W. Li Radiobiology Laboratory, Institute of Modern Physics, Chinese Academy of Sciences, 730000 Lanzhou, People’s Republic of China e-mail:

industrial interest for many years to produce microbial oils through oleaginous microorganisms that involve bacteria, yeasts, moulds and algae. Microbial polyunsaturated fatty acids, such as docosahexaenoic acid (DHA) and arachidonic acid (ARA), are very important in nutrition (Azeem et al. 1999; Ratledge 2004). Because of their similar composition of fatty acids to that of vegetable oils, microbial oils are now the potential feedstock for biodiesel production (Li et al. 2007; Zhu et al. 2008). Therefore any significant enhancement of the lipid yields in oleaginous strain will offer great opportunity for industrial production. Strain improvement has been achieved mainly through mutagen or genetic recombination in which the improved strains are randomly screened that result in low efficiency. The conventional methods to determine lipid contents in microorganisms typically require solvent extraction and weighing (Bligh and Dyer 1959; Gerhardt et al. 1994; Somashekar et al. 2001) in which the extraction often manipulates a significant amount of biological material and the process is very tedious and time-consuming. Attempts have been made to develop newer and better methods as evidenced by the reported measurement of absorbance in oleaginous yeast cells stained with Sudan Black B (Thakur et al. 1989; Patnayak and Sree 2005) and a colorimetric method based on the sulfo-phospho-vanillin reaction to quantify the lipids from bacteria samples (Izard and Limberger 2003). Although these methods allow a quick and direct estimation of intracellular lipid, developed methods are still in need. New and efficient isolation procedures that can significantly enhance the selection of the improved oleaginous strains would greatly speed up the industrial application of SCO. Fatty acid biosynthesis is catalyzed by type II fatty acid synthase (FAS) in most bacterium and by type I FAS in eukaryotes. One of the key features for mutant isolation is


In all experiments controls were sham-irradiated.0.34 g/l determined by conventional gravimetric method. Lanzhou. 123 . The absorption at 530 nm was measured using the distilled water as the control sample. isolated from the culture broth of the fungus Cephalosporium caerulens. we will present the development of an easy method that combines cerulenin incorporated medium for pre-selection and sulfo-phospho-vanillin reaction for estimation of the lipid yield to quickly select mutant strains after irradiating the oleaginous yeast cells with carbon ions. The irradiation doses were 5. Conventional methanol-chloroform extraction One gram of dried cells was hydrolyzed with 10 ml of 4 mol HCl in a boiling water bath for 1 h and then the sample was washed free of acid. calculated from particle fluencies and linear energy transfer (LET). MgSO4 Á 7H2O 1. The lipid concentration in the fermentation broth of pre-selected colonies was estimated from the reference curve.8 (v/v) were kept in the sample. the irradiated cultures were properly diluted and seeded on the YEPD agar incorporated with 8. pH is 5. Institute of Modern Physics. Based on the reports and our analyses. 15. methanol:chloroform:water at a ratio of 2:1:0. we have carried out a study of using cerulenin on the isolation and detection of mutants with high lipid yield in oleaginous microorganisms after mutagen treatment. and cooled for 5 min in a water bath at room temperature. Cerulenin [(2S)(3R)2. After mixing and 10 min incubation at 4°C. Pre-selection with cerulenin In order to determine the proper concentration of cerulenin for pre-selection. The layer of chloroform and lipid mixture was completely removed with a pipette. Materials and methods Microorganism and cultivation The red yeast Rhodotorula glutinis AY 91015 purchased from China Center for Type Culture Collection (CCTCC) was cultivated for 2–3 days at 28°C in YEPD medium (g/ l): glucose 20. To a test tube. Estimation of lipid concentration by sulfo-phospho-vanillin reaction Ten milliliters of fermentation broth was centrifuged.5 and KH2PO4 1. For more irradiation technical details please refer to the publication (Wang et al. the lipids were separated from the water-soluble material by diluting the sample with one volume of chloroform followed by one volume of water.0 (Origin Lab.5. 40 and 55 Gy. Finally.922 World J Microbiol Biotechnol (2009) 25:921–925 to identify the good inhibitors that form tight complexes in the enzyme active site so that only improved mutants with high fatty acid synthase activity can grow in the inhibitor incorporated medium. 100 ll cell suspension or 100 ll distilled water was added. peptone 10. After mixing with 2 ml of 18 mol sulfuric acid. Irradiation Exponential growing yeast cultures were irradiated with carbon ions of energy of 80 MeV/u at the heavy ion research facility of Lanzhou (HIRFL. All curves were plotted by Origin 7. Fermentation was performed in the modified YEPD medium with the following composition (g/l): glucose 30.96 lmol/l cerulenin for 5 days at 28°C. a very powerful mutation inducer. Five milliliters of phosphoric acid–vanillin (Sangon) reagent were added to each tube and incubated for 15 min at 37°C. Details for the preparation of the phosphoric acid–vanillin reagent are the same as described in the publication (Izard and Limberger 2003).09 to 0. peptone 10. A reference curve was obtained by plotting absorbance against the corresponding lipid concentration ranging from 0. 2001). the tubes were incubated in a boiling water bath for 10 min.24 to 11. Large colonies (diameter [ 2 mm) were picked out from the YEPD agar and transferred to fresh medium for preservation. 2240 lmol/l) at a concentration gradient from 2. According to the survival fraction and cerulenin concentration.. 2005). yeast extract 10 and agar 20 for solid medium. The cultures were grown in 100 ml media in 250 ml flasks for 6 days at 28 ± 1°C on a rotary shaker at 150 rev/min.20 lmol/l. yeast culture of the control sample was planted on the YEPD agar supplemented with cerulenin (Biomol. The survival fraction of the yeast cells after irradiation was determined by a standard colony formation assay. Afterwards. In this article. China). the chloroform in the extracted mixture was evaporated away and the total lipid was quantified by a gravimetric method. yeast extract 10. It had been reported that the content of intracellular polyunsaturated fatty acid produced in bacteria could be enhanced by cerulenin treatment (Morital et al. is a potent inhibitor because it targets specifically the b-ketoacyl-ACP synthases by becoming covalently attached to the active site cysteine so that type I and type II fatty acid synthase systems can be irreversibly inhibited (Heath et al. The yeast cell pellet was washed free of nutrients and resuspended in 2 ml of distilled water. America). 2008).3-epoxy-4oxo-7.10-dodecadienoylamide].

0 Absorbance The number of colonies represents the mean value of six independent samples Fig.10 0.96 lmol/l cerulenin is sufficient for the pre-selection of mutants with high fatty acid synthase activity. At a concentration of 8. Thus a 0. a linear equation y = 38.9 1. Following the assumed condensation reaction. Tietz 1982).2 Survival fraction 1. 1. 2. dehydration of an aldol-type intermediate is further assumed to yield a more highly unsaturated product that absorbs visible light (Tietz 1982).73 ± 0. while the colony size of the control sample without cerulenin treatment was pretty large (diameter [ 5 mm). and then the ketones condense with vanillin or a derivative of the vanillin under the influence of acid catalysis. The colorimetric method based on the sulfo-phosphovanillin reaction has been used for the determination of total serum lipids in humans (Frings and Dunn 1970. In general.995. As the result of visible large size.11 0.15 0. The relationship between the absorbance of stained cells and the lipid concentration of the yeast fermentation broth was fitted by least square.4 0. 2 The relationship between the absorbance of stained cells and the lipids concentration measured by the sulfo-phospho-vanillin method 123 . 33 large colonies (diameter [ 2 mm) were selected out from thousands of small colonies (diameter B 2 mm).08 0.1 Table 1 The survival fraction of Rhodotorula glutinis AY 91015 cells irradiated with 80 MeV/u carbon ions Dose (Gy) 0 (control) 5 15 40 55 Colonies 92 ± 9.5 19 ± 6. The absorbance was therefore measured at this wavelength. high mutation frequency is usually found at low survival.30 0. 1 Large colony formation efficiency of Rhodotorula glutinis AY 91015 grown on YEPD agar supplemented with cerulenin at a concentration gradient components of a lipid specimen first become oxidized to ketones. it was found that cerulenin suppressed efficiently the colony formation of Rhodotorula glutinis AY 91015.07 0. Only a few of possible mutants with enhanced lipid synthesis capacity grew into large ones that can be visibly isolated.05 0. The size of colony became smaller and smaller as the concentration of cerulenin in the culture medium increased. The maximum absorption of sulfo-phospho-vanillin stained yeast cells as well as lipid extracted from the yeasts was 530 nm.01 0 2 4 6 8 10 12 Cerulenin (µmol/l) Fig.02 0.6 8 ± 2.7 0.20 0.21 ± 0.05 0.2 0.World J Microbiol Biotechnol (2009) 25:921–925 923 1 Results After irradiation of the exponential growing culture of Rhodotorula glutinis AY 91015 to 80 MeV/u carbon ions. The results are listed in Table 1 and these measured data clearly show the cell survival fraction decreases with increasing irradiation dose. As shown in Fig. most of parent cells were prevented from growing into normal large colonies.67314 well fits the data with a correlation coefficient of 0.9 54 ± 4.2257x ? 0. Thus the pre-selection was performed mainly in yeast cultures irradiated with 40 and 55 Gy carbon ions.6 0.35 Lipid concentration (g/l) 0.00 ± 0.09 ± 0. even though the detailed chemical reactions remain unknown. After spreading the irradiated samples on the agar containing cerulenin and then incubating the culture at 28°C for 5 days.1 0.96 lmol/l nearly all colonies became very small (diameter B 2 mm).40 0. One assumption is that the unsaturated Large colony formation efficiency 0. During the determination of the proper concentration of cerulenin for pre-selection.8 0.8 67 ± 6. The relationship between large colony (diameter [ 2 mm) formation efficiency and cerulenin concentration incorporated in medium is shown in Fig.3 0.5 0.59 ± 0. Therefore YEPD agar supplemented with 8.25 0. the survival fraction of the yeast cells was determined by the colony formation assay.

it can be increased by selecting colonies with diameter [ 2 mm.6 Lipid concentration (g/l) sulfo-phospho-vanillin 0. 3.21 ± 0. As shown in Fig. With the advantage of easy handling.2 0. it can be expected that the use of cerulenin for isolation could be expanded into other oleaginous fungi after mutagen treatment.036 30. Based on the biomass of the dry weight.78 ± 3.2%. lipid concentrations in the fermentation broth from several experiments were measured by both methods. with increased lipid concentration compared with the control sample.34 g/l. The positive selection rate is 66%. as listed in Table 2.1 0. it does not require the breakage of cells involved in other chemical and enzymatic methods.65 g/l was outstanding in comparison with the control with lipid concentration of 0. the sulfo-phosphovanillin method is ideal to select mutants with high lipid yield in oleaginous yeast after the pre-selection with cerulenin.5 0.017 2. visible and labor saving isolation of improved mutant strains with high fatty acid synthase activity. the novel method developed by us has a number of advantages over others reported in literatures. Lipid content of the two mutants is substantially enhanced too. 1994).054 0. Among the improved 22 colonies. the analysis of the different ratios of lipid/DNA and lipid/protein can be used as an indicator of significant physiological conditions altering lipid. The higher lipid requires the fermentation sample be diluted to get a reasonable estimate.67%.34 ± 0. protein. 3 Comparison of lipid concentration measured by the sulfophospho-vanillin reaction and those determined by the conventional methanol-chloroform extraction method 123 . It has to be noted that the reference curve in Fig.03 ± 0. Furthermore. 28. Although the positive selection rate is of only 66%. M 5 and M 16 were 18.65 ± 0.17 ± 0.020 Lipid content (%) 18.028 0. Since the estimation of lipid concentration in the fermentation broth of the pre-selected colonies is based on the sulfo-phospho-vanillin reaction in the whole yeast cells. As it is confirmed in later experiments. All data are the means of three parallel samples Discussion The SCO production through oleaginous microorganisms usually starts from the selection of the improved strains with high lipid yield. 2 was drown with control yeast cells giving a relatively low lipid concentration in the fermentation medium (0. the positive selection rate could be 71% if colonies with diameter [ 3 mm were selected out.061 reference curve for estimation of the lipid concentration in the fermentation broth of the pre-selected colonies by sulfo-phospho-vanillin reaction was obtained. respectively. This may be due to the subjective criterion (diameter [ 2 mm) is not an optimum for the colony selection.78 and 30. such as thraustochytrids that are the potential polyunsaturated fatty acid producers for industrial use (Singh and Ward 1997). The pre-selection with cerulenin incorporated medium provides a rapid.1 0.3 0.3% and a maximum error of 5. of the total isolation of 33. The estimation of lipid concentration with the reference curve has shown that 22 colonies.3 0.924 World J Microbiol Biotechnol (2009) 25:921–925 Table 2 Biomass and lipid production in the control and mutant strains Strain Control M5 M 16 Biomass (g l-1) 2. To find out the correlation between the sulfo-phosphovanillin reaction and conventional methanol-chloroform extraction for lipid quantification. lipid concentration measured by the sulfo-phospho-vanillin reaction method are in very good agreement with those determined by the conventional method.60 ± 0.6 Lipid concentration (g/l) methanol-chloroform Fig.67 ± 2.938 Lipid concentration (g l-1) 0.34 g/l) while the lipid concentration of mutants with high lipid yield was surely higher than 0.022 2. As the sulfo-phospho-vanillin method can be used in conjunction with the quantitation of other cell components using the same sample.60 g/l and M 16 with lipid concentration of 0.4 0.19 ± 1. mutant M 5 with lipid concentration of 0.5 0.19.2 0. Considering our results and the report that cerulenin treatment is effective among certain kinds of eukaryotic microorganisms.281 28.4 0. calculations of the lipid yield of individual cell have shown that the lipid content of the control.34 g/l. As described here. no extraction and separation step is necessary and only a small amount of biological material is required while the typical extraction of lipids uses a two-phase separation that requires a significant amount of biological material as well as a significant amount of manipulation of the biological material preceding the quantitation (Gerhardt et al. or DNA synthesis in the mutant strain when 0. giving an average error of 2.

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