Journal of Biotechnology 168 (2013) 303–314

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Importance of the methyl-citrate cycle on glycerol metabolism in the yeast Yarrowia lipolytica Seraphim Papanikolaou a,∗ , Athanasios Beopoulos b , Anna Koletti a , France Thevenieau b , Apostolis A. Koutinas a , Jean-Marc Nicaud c , George Aggelis d,e a

Department of Food Science and Human Nutrition, Agricultural University of Athens, 75 Iera Odos, Athens 11855, Greece INRA, UMR1319, Micalis, F-78352, Jouy-en-Josas, France c CNRS, Micalis, F-78352, Jouy-en-Josas, France d Unit of Microbiology, Department of Biology, Division of Genetics, Cell and Development Biology, University of Patras, Patras 26500, Greece e Department of Biological Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia b

a r t i c l e

i n f o

Article history: Received 14 August 2013 Received in revised form 7 October 2013 Accepted 14 October 2013 Available online 25 October 2013 Keywords: Yarrowia lipolytica Oleaginous yeast Citric acid Crude glycerol Methylcitrate cycle

a b s t r a c t A novel approach to trigger lipid accumulation and/or citrate production in vivo through the inactivation of the 2-methyl-citrate dehydratase in Yarrowia lipolytica was developed. In nitrogen-limited cultures with biodiesel-derived glycerol utilized as substrate, the phd1 mutant (JMY1203) produced 57.7 g/L of total citrate, 1.6-fold more than the wild-type strain, with a concomitant glycerol to citrate yield of 0.91 g/g. Storage lipid in cells increased at the early growth stages, suggesting that inactivation of the 2-methylcitrate dehydratase would mimic nitrogen limitation. Thus, a trial of JMY1203 strain was performed with glycerol under nitrogen-excess conditions. Compared with the equivalent nitrogen-limited culture, significant quantities of lipid (up to ∼31% w/w in dry weight, 1.6-fold higher than the nitrogen-limited experiment) were produced. Also, non-negligible quantities of citric acid (up to ∼26 g/L, though 0.57-fold lower than the nitrogen-limited experiment) were produced, despite remarkable nitrogen presence into the medium, indicating the construction of phenotype that constitutively accumulated lipid and secreted citrate in Y. lipolytica during growth on waste glycerol utilized as substrate. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The current exploitation of biodiesel has resulted in a significant glut of crude glycerol on the market. Large quantities of glycerolcontaining water are also generated by bioethanol and alcoholic beverage production units as well as by fat saponification facilities (Papanikolaou et al., 2000). Thus, conversion of this low-cost material to higher added-value compounds (e.g. 1,3-propanediol, 2,3-butanediol, ethanol, mannitol, acetic acid, etc.) by the means of fermentation technology is attracting substantial and continuously increasing interest (Chatzifragkou et al., 2011; Metsoviti et al., 2012, 2013; Papanikolaou et al., 2000, 2008). A recently developed approach related with the fermentation of glycerol refers to its conversion into citric acid and/or microbial lipids (single cell oil–SCO) by the oleaginous yeast Yarrowia lipolytica (André et al., 2009; Fontanille et al., 2012; Imandi et al., 2007; Kamzolova et al., 2011; Levinson et al., 2007; Makri et al., 2010; Morgunov et al., 2013;

∗ Corresponding author at: Laboratory of Food Microbiology and Biotechnology, Agricultural University of Athens, Athens, Greece. Tel.: +30 210 5294700; fax: +30 210 5294700. E-mail address: [email protected] (S. Papanikolaou). 0168-1656/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2013.10.025

Papanikolaou and Aggelis, 2002, 2009; Papanikolaou et al., 2002a, ´ 2008; Rymowicz et al., 2006, 2009; Rywinska and Rymowicz, 2010). Y. lipolytica is a promising candidate for biotechnological applications because it is amenable of accumulating lipids from a variety of hydrophobic substances (e.g. free-fatty acids, triacylglycerols and n-alkanes, through the ex novo lipid synthesis process; for reviews see: Beopoulos et al., 2009a,b; Fickers et al., 2005; Papanikolaou and Aggelis, 2011a,b), with the multi-gene families encoding for key enzymes involved in hydrophobic substrates breakdown (e.g. acyl-CoA oxidases, lipases) having been studied in details (Beopoulos et al., 2009a,b; Fickers et al., 2005). Furthermore, genetically modified Y. lipolytica strains with improved lipid accumulation capacity have been obtained through genetic engineering of the lipid metabolism, when both sugars and/or hydrophobic materials have been used as substrates (Beopoulos et al., 2008, 2012; Dulermo and Nicaud, 2011; Mliˇcková et al., 2004a,b; Tai and Stephanopoulos, 2013). Biosynthesis of both citric acid and SCO from carbon sources, including glucose and glycerol, is triggered by the depletion of an essential nutriment, and in particular nitrogen, from the culture medium (Beopoulos et al., 2009a; Papanikolaou and Aggelis, 2009). Upon nitrogen limitation and in the presence of excess carbon, oleaginous microorganisms produce large amounts of TCA cycle intermediates, including citric

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acid and iso-citric acid, which are not further catabolized. Nitrogen exhaustion results in a rapid decrease of the intra-cellular AMP (adenosine monophosphate) concentration, as AMP is cleaved to produce NH4 + ions, indispensable for cell growth. This results in the inactivation of iso-citrate dehydrogenase in the TCA cycle and, consequently, the accumulation of both iso-citric and citric acids into the mitochondria. When the intra-mitochondrial citric acid concentration reaches a critical value, citrate enters the cytoplasm in exchange for malate. Citric acid is then cleaved by ATP-citrate lyase (ACL), the key enzyme of the lipid accumulation process in oleaginous microorganisms, to yield acetyl-CoA and oxaloacetate. The resulting acetyl-CoA is carboxylated by acetyl-CoA carboxylase (ACC1) to form malonyl-CoA, the substrate for the biosynthesis of acyl-CoA esters and subsequently triacylglycerols (for intermediate metabolic pathways in oleaginous microorganisms, see Fig. 1; for critical reviews see: Beopoulos et al., 2009b; Papanikolaou and Aggelis, 2011a,b; Ratledge and Wynn, 2002). As indicated, biosynthesis of both citric acid and SCO from glycerol or related compounds utilized as the sole substrate in their initial steps is biochemically equivalent and performed after nitrogen depletion from the culture medium. The production of citric acid and/or SCO during growth of Y. lipolytica on glycerol or similarly metabolized compounds (e.g. glucose or other sugars—with utilization, thus, of the de novo fatty acid biosynthesis pathway) seems strain dependent, since in some cases in flask or bioreactor operations predominantly SCO is produced [lipid in dry cell weight (DCW) >35%; see: Papanikolaou and Aggelis, 2002; Tsigie et al., 2011, 2012; Fontanille et al., 2012] whereas in other studies citric acid (and potentially sugar-alcohols) is secreted into the growth medium with relatively low quantities of lipid (e.g. 40% (v/v), incubation temperature T = 28 ◦ C. Each point is the mean value of two independent measurements (SD≤10%). Strain

Carbon source

Time (h)

Glolcons (g/L)

Glccons (g/L)

X (g/L)

Lipid (%, w/w)

L (g/L)

YL/Glol (YL/Glc ) (g/g)

Cit (g/L)

YCit/Glol (YCit/Glc ) (g/g)

W29

Glca

91.5 170 72.5 150 72 160 96 160

– – – – 12.9 39.9 19.0 39.9

21.9 37.0 12.2 34.5 – – – –

11.2 11.0 3.2 1.8 10.7 12.5 5.5 7.0

5.8 2.4 10.1 5.1 10.0 1.6 14.9 10.0

0.65 0.26 0.32 0.09 1.07 0.20 0.82 0.70

0.030 0.007 0.026 0.003 0.083 0.005 0.043 0.018

18.2 31.4 6.8 15.2 6.0 19.1 11.8 31.0

0.83 0.85 0.56 0.44 0.47 0.48 0.62 0.78

b

JMY1203

Glca b

W29

Glola b

JMY1203

Glola b

a b

Representations when maximum quantity of lipid in dry weight (%, w/w) was achieved Representations when maximum concentration of total citric acid (in g/L) was achieved

accumulation in media containing various concentrations of crude glycerol. 3.3. Growth of Yarrowia lipolytica strains on crude glycerol at elevated initial substrate concentrations under nitrogen-limited conditions In the second series of trials, nitrogen-limited media containing the same initial nitrogen quantity as previously were employed, whereas crude glycerol was utilized at elevated initial concentrations (Glol0 at 60 and 90 g/L) (Table 3). Both strains consumed high quantities of the employed substrate, whereas, as previously, W29 strain presented a higher biomass concentration compared with JMY1203. Increment of Glol0 concentration resulted in some decrease of DCW quantity produced by W29 (see Tables 2 and 3) suggesting potential substrate inhibition. A similar observation can be made also for the strain JMY1203, taking into consideration that DCW quantity was ∼7 g/L for the trial with Glol0 = 40 g/L, decreasing to ∼4 g/L for the experiments with elevated Glol0 concentrations (see Tables 2 and 3). Decrease of biomass concentration as the culture proceeded was observed for the experiment with Glol0 = 90 g/L, principally for W29 strain and to lesser extent for JMY1203 strain. One the other hand, Cit concentration (in g/L) noticeably increased with the increment of Glol0 concentration into the medium; W29 strain produced ∼2-fold Cit quantities at Glol0 = 90 g/L when compared to those produced at Glol0 = 40 g/L (see Tables 2 and 3). In any case, the conversion yield of citric acid produced per glycerol consumed (YCit/Glol ) was ∼0.43–0.48 g/g (see Tables 2 and 3), suggesting that the conversion threshold of glycerol into citric acid by the above-mentioned strain could not be higher than 0.48 g/g, under the given culture conditions. On the other hand, for the strain JMY1203, even though the DCW obtained was substantially lower than that with W29, both Cit production and the glycerol to citrate conversion yield increased with glycerol concentration. The amount of citrate produced rose from 31 g/L at Glol0 = 40 g/L to nearly 58 g/L at Glol0 = 90 g/L and the yield of the conversion of glycerol into citric acid (YCit/Glol value) increased from 0.78 g/g to the extremely high value of 0.91 g/g. These values represent a 1.6-fold greater citrate production and 2.1-fold higher glycerol to citrate conversion yield than for W29. Citric acid was the main compound of total citrate produced, since the iso-citric acid assay demonstrated that iso-citric acid was ∼5–8%, w/w, of total citric acid produced regardless of the tested strain and the Glol0 concentration into the medium (data not presented). Moreover, HPLC analysis of the culture liquid showed that no other metabolic compounds relevant to the TCA or methyl-citrate cycle (e.g. acetic acid, pyruvic acid, propionic acid, methyl-citric acid, etc.) or other low-molecular weight compounds relevant to the metabolism of Y. lipolytica (e.g. mannitol, erythritol) were identified

for both the wild and the mutant strain (the detection threshold of these compounds is ∼0.2–0.4 g/L). In the trial with the Citmax quantity achieved, the quantity of iso-citric acid found was ∼5%, w/w, of Cit. JMY1203 strain virtually accumulated significant quantities of lipids at Glol0 = 90 g/L (∼27% in DCW). The respective value for the wild-type W29 strain was 11.1%. Representative kinetics of both W29 and JMY1203 strains (case of Glol0 = 90 g/L) are shown in Fig. 2 a–d. Extra-cellular nitrogen (initial NH4+ at 55 ± 10 mg/L) was exhausted around 60 h after inoculation for both strains (Fig. 2a). Nitrogen uptake rate was similar regardless of the Glol0 concentration employed or the utilization of glucose as substrate for both tested strains (kinetics not presented). Despite nitrogen exhaustion, biomass concentration clearly increased for the case of the strain W29, reaching to Xmax value ∼10 g/L after 150 h (Fig. 1b), while no accumulation of lipid was observed (see Table 3). These facts indicate that potentially other storage compounds (e.g. polysaccharides) were accumulated inside the yeast cell. On the other hand, biomass production for the strain JMY1203 was clearly suppressed after the exhaustion of assimilable nitrogen from the culture medium, while even some biomass decrease was observed after imposition of nitrogen limitation into the medium (Fig. 2a and b). Similarly, concerning biomass evolution for the strain W29, after maximum concentration had been achieved, DCW presented a remarkable decrease in its value. In any case and despite biomass concentration decrease, cell population was active, since glycerol assimilation and Cit production occurred for both strains during biomass decrease phase (Fig. 2b–d). Glycerol consumption was constant and almost linear for the two strains tested throughout the culture, in both nitrogen nonlimited (0–60 h) and nitrogen-limited (60–330 h) growth phases (Fig. 2c). Glycerol consumption rate of W29, rGlol (expressed as rGlol = − Gol/t), was 0.26 g/L.h, higher than 0.18 g/L.h, which obtained for JMY1203. Likewise, citric acid production for both strains seemed constant and linear; citric acid production rate (rCit , expressed as rCit = − Cit/t) was 0.11 g/L.h for the strain W29 whereas it increased by 1.54-fold for JMY1203 (0.17 g/L.h) (Fig. 2d). The behavior of each strain was the same at all initial glycerol concentrations tested, indicating that glycerol consumption and citrate production rates are strain dependent. In any case the high Cit production (in g/L) together with the exceptional YCit/Glol value obtained, indicate the potentiality of the utilization of the JMY1203 strain towards the conversion of crude glycerol into citric acid. Lipid accumulation patterns differed substantially between the two strains (Figs. 3a and b). During the non-limiting nitrogen phase, W29 accumulated lipids at a rate independent of the glycerol concentration, reaching a maximum of ∼10% of CDW in all conditions tested. However, once nitrogen was depleted, the lipids were readily remobilized at low glycerol concentrations, but not at high glycerol concentrations (Fig. 3a). This suggests that lipid

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Table 3 Quantitative data of Yarrowia lipolytica strains JMY1203 and W29 originated from cultures on glycerol-based nitrogen-limited media at two initial glycerol concentrations (Glol0 , g/L) and constant initial nitrogen concentration. Representation of biomass (X, g/L), lipid (L, g/L), total citric acid (Cit, g/L) and consumed glycerol (Glolcons , g/L) when: maximum quantity of lipid in dry microbial mass (%, w/w) (a) and the maximum concentration of total citric acid (b) were achieved. Culture conditions: growth on 250-mL flasks at 180 rpm, Glol0 = 60 and 90 g/L, initial pH = 6.0 ± 0.1, pH ranging between 4.8 and 6.0, DOT > 40% (v/v), incubation temperature T = 28 ◦ C. Each point is the mean value of two independent measurements (SD≤10%). Strain

Glol0 (g/L)

Time (h)

Glolcons (g/L)

X (g/L)

Lipid (%, w/w)

L (g/L)

YL/Glol (g/g)

Cit (g/L)

YCit/Glol (g/g)

W29

60a

72 226 72.5 281 96 310 78 340

19.7 59.9 16.0 60.1 24.0 85.5 14.4 63.2

7.6 10.7 3.1 3.9 8.0 5.3 4.5 3.5

9.5 4.1 19.0 10.5 11.1 8.6 26.6 6.9

0.73 0.44 0.59 0.41 0.88 0.46 1.20 0.24

0.037 0.007 0.031 0.007 0.037 0.005 0.083 0.004

1.9 27.0 4.1 45.5 11.4 36.8 9.1 57.7

0.10 0.45 0.26 0.76 0.47 0.43 0.64 0.91

b

JMY1203

60a b

W29

90 a b

JMY1203

90 a b

a b

Representations when maximum quantity of lipid in dry weight (%, w/w) was achieved. Representations when maximum concentration of total citric acid (in g/L) was achieved.

turnover pathway is potentially regulated by the available glycerol concentration into the culture medium. In contrast, lipid accumulation by the strain JMY1203 was clearly dependent on the glycerol concentration during the non-limiting nitrogen phase, whereas lipid turnover during the nitrogen-limiting phase was not greatly affected by the glycerol concentration (Fig. 3b). In the strain JMY1203, lipid was accumulated up to 26.6% of CDW when Glol0 was 90 g/L, while the respective value was 14.9% when Glol0 was 40 g/L. For both strains (W29 and JMY1203) the kinetic results showed that lipid in DCW increased and, therefore, de novo lipid accumulation occurred even though quantities of assimilable nitrogen were found into the culture medium (this observation was much clearer for the strain JMY1203) (see Figs. 2a, 3a and b).

Finally, concerning DOT evolution, once nitrogen was depleted, DOT (pO2 ) values decreased, and then increased as the culture proceeded (kinetics not presented). As nitrogen starvation became a dominant feature of the cultures, intra-cellular lipids were degraded and large quantities of citric acid accumulated in the culture medium, with a concomitant rise in the DOT value. 3.4. Growth of Yarrowia lipolytica strains on crude glycerol under carbon-limited conditions Given the kinetic behavior of the mutant strain observed in the previous trials (e.g. accumulation of non-negligible lipid quantities in DCW at the early stages of the nitrogen-limited cultures, in which virtually nitrogen was found into the medium), it was

Fig. 2. Kinetics of ammonium ion consumption (a), biomass production (b), glycerol consumption (c) and total citric acid production (d) during growth of Yarrowia lipolytica strains W29 and JMY1203 in nitrogen-limited glycerol-based media. Culture conditions: growth in 250-mL flasks at 185 rpm, Glol0 = 90 g/L, initial pH = 6.0 ± 0.1, pH subsequently between 4.8 and 6.0, DOT > 40% (v/v), incubation temperature T = 28 ◦ C. Each point is the mean value of two independent measurements.

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Table 4 Quantitative data of Yarrowia lipolytica strains JMY1203 and W29 originated from cultures on glycerol-based carbon-limited media at initial glycerol concentration (Glol0 , g/L) of 60 g/L. Representation of biomass (X, g/L), lipid (L, g/L), total citric acid (Cit, g/L) and consumed glycerol (Glolcons , g/L) when: maximum quantity of lipid in dry microbial mass (%, w/w) (a) and the maximum concentration of total citric acid (b) were achieved. Culture conditions: growth on 250-mL flasks at 180 rpm, Glol0 = 60 g/L, initial pH = 6.0 ± 0.1, pH ranging between 4.8 and 6.0, DOT > 40% (v/v), incubation temperature T = 28 ◦ C, NH4 + concentration at the end of the trials at ∼300 mg/L. Each point is the mean value of two independent measurements (SD≤10%). Strain

Glol0 (g/L)

Time (h)

Glolcons (g/L)

X (g/L)

Lipid (%, w/w)

L (g/L)

YL/Glol (g/g)

Cit (g/L)

YCit/Glol (g/g)

W29

60a

68 168 68 168

30.3 55.5 27.5 55.9

7.5 15.1 3.2 3.6

9.9 6.0 30.7 22.5

0.74 0.91 0.98 0.81

0.025 0.016 0.035 0.014

1.0 4.1 5.8 25.8

0.03 0.07 0.21 0.46

b

60 a

JMY1203

b

a

Lipid (%, w/w), Glol =90 g/L

20

0

Lipid (%, w/w), Glol =60 g/L 0

Lipid in dry weight (%, w/w)

Lipid (%, w/w), Glol =40 g/L 0

15

10

5

0 0

b

50

100

150 200 Time (h)

250

300

350

Lipid (%, w/w), Glol =90 g/L 0

40

Lipid (%, w/w), Glol =60 g/L 0

35

Lipid (%, w/w), Glol =40 g/L

Lipid in dry weight (%, w/w)

0

30 25 20 15 10 5 0 0

50

100

150 200 Time (h)

250

300

350

Fig. 3. Kinetics of total lipid content as a percentage of dry biomass (%, w/w) during growth of Yarrowia lipolytica strains W29 (a) and JMY1203 (b) in nitrogen-limited glycerol-based media. Culture conditions: growth in 250-mL flasks at 180 rpm, Glol0 = 40, 60 and 90 g/L, initial pH = 6.0 ± 0.1, pH subsequently between 4.8 and 6.0, DOT > 40% (v/v), incubation temperature T = 28 ◦ C. Each point is the mean value of two independent measurements.

important to investigate the physiological behavior of the strain in trials with glycerol employed as the sole carbon source at elevated initial concentrations, and nitrogen being found in excess into the medium throughout the culture. Therefore, an experiment with Glol0 = 60 g/L and ammonium sulfate and yeast extract at concentrations of 5.0 g/L and 2.5 g/L, respectively, was performed, with the results for both the JMY1203 and the W29 strains being presented in Table 4. From the obtained results it can be seen that again, much higher quantities of DCW were obtained for the wild compared with the mutant strain. Moreover, as it was expected, growth of the

wild-type W29 strain under carbon-limited conditions compared with the equivalent nitrogen-limited trial at Glol0 = 60 g/L resulted in much higher maximum DCW quantities, more rapid glycerol consumption and drastically lower Cit quantities detected into the culture medium (see Tables 3 and 4). In fact, whilst the Citmax quantity obtained for the W29 strain under nitrogen-limited conditions was 27 g/L, only 4.1 g/L of citric acid were synthesized when growth was performed under carbon-limited conditions with similar Glol0 concentrations employed in both cases. It is also noted that the HPLC analysis on the culture medium performed in the carbonlimited experiment for the W29 strain indicated that besides this small quantity of citric acid (which gradually appeared with the culture time), no other metabolic compounds (e.g. acetic acid, pyruvic acid, mannitol, erythritol, etc) were identified at the end of culture (the detection threshold of these compounds is ∼0.2–0.4 g/L). Quite interesting results were achieved for the phd1 mutant strain cultivated on crude glycerol (at Glol0 = 60 g/L) under carbon-limited conditions; while the maximum DCW concentration recorder was similar between the carbon- and nitrogen-limited cultures with Glol0 = 60 g/L (Xmax values between 3.2 and 4.2 g/L), remarkable quantities of lipid (e.g. lipid in DCW of ∼31%) were produced, even at the early steps of the growth. Maximum lipids in DCW were quite higher than the respective nitrogen-limited trial (1.61-fold higher), while as in the previous trials, cellular lipids in DCW seemed to somehow decrease with the time, whereas simultaneously nonnegligible quantities of citric acid were secreted into the medium. Citmax quantity achieved though in the carbon-limited trial was 0.57-fold lower than the respective nitrogen-limited experiment with Glol0 = 60 g/L (25.8 against 45.5 g/L—see Tables 3 and 4). As previously, no other extra-cellular compounds (e.g. acetic acid, propionic acid, mannitol, etc.) were identified into the culture medium. In any case however, it must be pointed out that noteworthy quantities of both storage lipids and citric acid were constitutively produced by the JMY1203 mutant, whereas remarkable quantities of nitrogen remained unconsumed at the end of the culture (initial NH4 + concentration at ∼900 mg/L, NH4 + concentration at the end of the trials at ∼300 mg/L, kinetics not presented). Comparison of the kinetic profiles between the carbon- and the nitrogen-limited trial with Glol0 = 60 g/L demonstrated that glycerol was much more rapidly consumed in the former than in the later case (see Fig. 4a), whereas much higher quantities of lipid in DCW were produced in the carbon-limited experiment even at the early growth steps compared with the nitrogen-limited experiment (see Fig. 4b), indicating the constitutive phenotype constructed as regards the process of de novo lipid accumulation in the phd1 mutant. Finally, non-negligible Cit quantities were secreted by the mutant strains even under nitrogen-excess conditions, with rCit being almost identical for the carbon- and the nitrogen-limited experiment. 3.5. Microbial lipid analysis The fatty acid (FA) composition of cellular lipid produced was studied at the late growth phases for both strains cultivated on glucose and crude glycerol is depicted in Table 5. From the obtained

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Fig. 4. Kinetics of glycerol consumption (a), total lipid content as a percentage of dry biomass (%, w/w) (b) and total citric acid production (c) during growth of Yarrowia lipolytica strain JMY1203 in carbon- and nitrogen-limited glycerol-based media. Culture conditions: growth in 250-mL flasks at 180 rpm, Glol0 = 60 g/L, initial pH = 6.0 ± 0.1, pH subsequently between 4.8 and 6.0, DOT > 40% (v/v), incubation temperature T = 28 ◦ C. Each point is the mean value of two independent measurements.

results it can be observed that FA composition changed as function of the Glol0 concentration employed and the culture time, while equally differences were observed between the growth on glucose and glycerol. Likewise, some differences in the FA profiles were observed between both employed strains, since cultivation of W29 strain resulted in the synthesis of a microbial lipid less rich in oleic acid (9 C18:1) and richer in linoleic acid (9,12 C18:2) compared with JMY1203. Moreover, for the equivalent nitrogen-limited

trials on Glol and Glc (initial concentration 40 g/L) FA composition in the cellular lipids of the strain W29 presented some remarkable differences, since the cultivation on glucose was accompanied by synthesis of a lipid more rich in 9,12 C18:2. Equally, remarkable differences in the FA composition of cellular lipids were observed for the strain JMY1203, for the similar trials on Glol and Glc; growth on glucose was accompanied by the synthesis of a lipid richer in 9 C18:1 and 9,12 C18:2 and less rich in saturated FAs.

Table 5 Fatty acid profile of the wild-type (W29) and the mutant (JMY1203) strain as percentage of total fatty acids. Strains were grown in nitrogen-limited media containing glucose (at 40 g/L) or glycerol (at 40, 60 and 90 g/L) and carbon-limited media containing glycerol (at 60 g/L) (SD≤10%). Culture conditions as in Tables 2, 3 and 4. Fatty acids (%, w/w) Substrate

Glc0 = 40 g/L Glol0 = 40 g/L Glol0 = 60 g/L Glol0 = 90 g/L Glol0 = 60 g/Lc a

Growth phase

a

LE or ES Sb LE or ES S LE or ES S LE or ES S LE or ES S

9

C16:0

9

C18:0

9,12

C18:1

C18:2

W29

1203

W29

1203

W29

1203

W29

1203

W29

1203

12.7 19.2 17.8 22.3 21.5 20.1 15.9 16.8 19.1 17.2

9.7 13.1 25.7 21.5 24.0 21.2 22.7 17.3 22.9 20.2

6.2 6.9 9.6 14.1 6.9 8.4 6.0 6.8 5.9 6.1

6.2 9.1 4.4 8.1 3.8 5.8 5.0 7.1 2.9 5.0

5.3 8.1 15.1 9.0 18.1 19.5 15.5 14.5 15.2 12.1

7.0 2.2 10.0 13.3 15.2 14.7 13.4 19.1 12.9 11.5

55.1 44.6 54.2 48.1 44.9 40.8 47.1 46.7 48.1 47.5

64.8 58.7 54.2 47.6 52.4 52.0 59.7 52.3 55.2 54.9

20.4 20.9 2.8 6.2 9.9 11.2 9.1 14.9 11.0 12.9

11.8 16.4 5.1 9.1 4.1 5.8 4.0 3.2 3.9 5.0

LE Late exponential phase and ES: Early stationary phase (60–90 h). S Stationary phase (110–160 h). c Nitrogen-excess fermentation. b

C16:1

S. Papanikolaou et al. / Journal of Biotechnology 168 (2013) 303–314 Table 6 Distribution of lipid fractions and fatty acid composition of total lipids (T), neutral lipids (N), glycolipids plus sphingolipids (G + S) and phospholipids (P) of Yarrowia lipolytica strains W29 and JMY1203 during growth in nitrogen-limited media containing glycerol (at 90 g/L). Culture conditions as in Table 4, sampling point in which lipids were analyzed was at the stationary growth phase (110–160 h) (SD≤10%). Strain

Lipid fraction

W29

T N G+S P T N G+S P

JMY1203

%, w/w 73.0 18.9 8.1 91.0 2.2 6.8

16:0

9

16.8 15.9 18.1 15.5 17.3 17.0 17.6 15.9

6.8 7.0 6.1 6.2 7.1 6.1 7.4 7.8

16:1

18:0

9

14.5 12.8 15.1 13.9 19.1 20.4 18.1 15.4

46.7 48.1 45.1 45.1 52.3 52.0 50.2 53.1

18:1

9,12

18:2

14.9 13.1 14.1 17.9 3.2 2.9 3.3 7.1

Additionally, for the strain W29 increment of Glol0 concentration resulted in a slight decrease in the concentration of 9 C18:1 and a more clear increase in the concentration of 9,12 C18:2, while the opposite trend was found for the strain JMY1203. On the other hand, not any remarkable differences in the FA composition of both strains were observed between the equivalent carbon- and nitrogen-limited trials. Finally, as a general observation it must be pointed out that the cellular 9 C18:1 concentration presented the tendency to decrease with evolution of the culture. Analysis of the various lipid fractions (N, G + S and P fractions) for Glol0 = 90 g/L at the stationary phase, showed that the neutral lipid fraction of JMY1203 contained 1.2-fold more lipids than that of the W29 strain associated with, mostly, less in the G + S fraction (Table 6). FA composition of N fraction showed similarities with that of total lipids, whereas in general the fraction of P was slightly more rich in poly-unsaturated FA (principally 9,12 C18:2) (Table 6). Potentially, the differences in the quantities of N, G + S and P for the strains W29 and JMY1203 can reflect in the differentiations in the FA composition observed of the total lipids of these microorganisms (see Tables 5 and 6); in the trials with lower quantities of total lipids produced (case of W29 strain), higher quantities of G + S and P fractions (in %, w/w, in total lipids), which are more unsaturated, were synthesized. Therefore, it should be the higher quantity of N fraction (that is slightly more saturated compared with the G + S and P fractions) that should bring about the fact that globally, the total lipids of JMY1203 were more rich in the FA 9 C18:1 and less rich in the FA 9,12 C18:2 than the ones of W29. 4. Discussion In the oleaginous microorganisms, in conditions of nitrogen limitation and in the presence of excess carbon found in the form of glucose, glycerol or similarly metabolized compounds, after nitrogen depletion, preferential channeling of the carbon flux towards lipid synthesis is performed. Due to nitrogen exhaustion, a rapid decrease of the concentration of intra-cellular AMP (adenosine monophosphate) occurs, since the microorganism cleaves the available AMP in order to gain NH4 + ions, indispensable for its maintenance. The excessive decrease of intra-cellular AMP concentration blocks the TCA cycle function, and thus, iso-citric and, finally, citric acid is accumulated inside the mitochondrion. Thereafter, citrate enters the cytoplasm in exchange with malate, with citric acid being cleaved by ATP-citrate lyase (ACL), the enzyme-key of lipid accumulation process in the oleaginous microorganisms, to yield acetyl-CoA and oxaloacetate. The synthesized acetyl-CoA is carboxylated by acetyl-CoA carboxylase (ACC) to provide the malonyl-CoA substrate for the biosynthesis of acyl-CoA esters and subsequently the triacylglycerols (for critical reviews see: Beopoulos et al., 2009a; Papanikolaou and Aggelis, 2011a,b—see intermediate metabolism scheme of oleaginous microorganisms in Fig. 1).

311

Carbon excess in the anabolism of the yeast Y. lipolytica can, besides the production of storage lipids, also be triggered towards the secretion into the growth medium of low-molecular weight metabolites (citric acid and to lesser extent acetic acid or polyols) (André et al., 2009; Chatzifragkou et al., 2011; Papanikolaou et al., 2002a, 2009). On the other hand, if the extra-cellular carbon supply is exhausted, stored lipids may be mobilized. In general, in Y. lipolytica, three metabolic states can be defined as a function of the C/N ratio for a constant nitrogen flux: when the carbon flux is lower than that required for growth, cells mobilize their storage lipids; if the carbon influx is optimal, the growth rate is maximum (biomass production); if carbon is present in excess and nitrogen is limiting, metabolism switches towards citric acid and/or lipid production. In some cases, the concentration of available nitrogen was considered of importance; critical nitrogen concentration for induction of lipid production has been found to be ∼10−3 mol/L. When nitrogen concentration dropped below this threshold value, secondary metabolites, and notably citric acid, were produced (Beopoulos et al., 2009a; Cescut, 2009). In other cases, growth of Y. lipolytica in nitrogen-limited glucose- or glycerol-based media resulted in sequential production of intra-cellular lipid and extracellular citric acid; in the first steps of nitrogen limitation, lipid accumulation was triggered and thereafter lipid content decreased with time, even though significant substrate quantities remained unconsumed into the medium. The period of intra-cellular lipid degradation (turnover) coincided with the secretion of citric acid in non-negligible quantities into the culture medium (Papanikolaou et al., 2009; Makri et al., 2010). The findings in the current investigation are entirely consistent with this observation, regardless of the quantity of lipid (in %, w/w) found inside the cells (see Figs. 3a and b, 4b). Nevertheless, intra-cellular lipid breakdown, presumably performed via ˇ-oxidation, was much clearer in the case of JMY1203. Though, in other studies, lipid content increased constantly (from 7.4% to 17.8%, w/w) over the whole range of the batch fermentation, with significant simultaneous citric acid production (Rymowicz et al., 2010). The above-mentioned complex regulation makes it difficult in many instances to obtain high rates of lipid accumulation in batch culture, where the nitrogen concentration cannot be maintained, and therefore metabolic switching is not controlled. In such conditions, lipid accumulation and citric acid production occur simultaneously, resulting in only moderate lipid accumulation. Indeed, under nitrogen-limited conditions in flasks, only limited quantities of cellular lipids (e.g. lipid in DCW 48%) on both defatted rice bran hydrolysate and sugarcane bagasse hydrolysate in shake flasks (Tsigie et al., 2011, 2012). Also Y. lipolytica MUCL 28849 cultivated on glucose or glycerol in batch bioreactor experiments produced significant quantities of lipid (∼37% w/w, lipid yield ∼16 g/L) (Fontanille et al., 2012). In other cases, in fed-batch bioreactor experiments, the amount of single-cell oil can reach 40% of CDW (Beopoulos et al., 2009a), even without citric acid

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production by fine-tuning of the glucose feeding (Cescut, 2009). ´ Furthermore, according to Rywinska et al. (2013) and Morgunov et al. (2013) air saturation of the culture medium seems to be one of the major determining factors responsible for citric acid production by Y. lipolytica strains. Recently, genetic engineering approaches have been used to increase lipid accumulation in Y. lipolytica, either by (i) increasing glycerol-3-phosphate (G3P) levels through inactivation of the glycerol-3-phosphate dehydrogenase (GUT2) or overexpression of the glycerol-3-phosphate dehydrogenase (GPD1) isomer, catalyzing the reverse reaction (Beopoulos et al., 2008; Dulermo and Nicaud, 2011); (ii) overexpression of DAG acyltransferases (DGA1, DGA2) (Beopoulos et al., 2012); (iii) disruption of the MIG1 gene alleviating the repression of several genes by glucose (47.8% lipids vs 36% for the wild type) (Wang et al., 2013); and (iv) simultaneously increasing fatty acid and TAG synthesis by over-expression of ACC1 and DGA1. Lipid accumulation in the strain overproducing ACC1 and DGA1 reached 41.4% of CDW in shake-flask experiments versus 11.7% of CDW for the wild-type strain (Tai and Stephanopoulos, 2013). Alternatively, the carbon flux can be oriented towards citrate production by inactivation of the ATP citrate lyase (ACL1): this results in the abolition of citrate conversion to acetyl-CoA and overexpression of isocitrate lyase (ICL1) leading to high citric acid production (Liu et al., 2013). In this study, we developed a novel approach to trigger lipid accumulation and/or citrate production. Rather than using nitrogen limitation, the approach was based on the inhibition of the TCA cycle by inactivation of the PHD1 gene. This modification apparently blocked the TCA cycle at the aconitate level, as the accumulated 2methyl-citrate inhibits aconitase and probably also the transport of citrate into mitochondria, resulting in the secretion of citrate. Therefore, despite remarkable nitrogen excess into the medium, the mutant strain constitutively favored lipid accumulation and citric acid production over biomass production (see Table 4). Interestingly, much higher lipid in DCW quantities were produced by the mutant strain under carbon- rather than under nitrogen-limited conditions (see Fig. 4b) while if nitrogen limitation was imposed JMY1203 metabolism switched mostly towards citrate secretion (see Tables 3 and 4). In any case, the construction of a phenotype capable to produce constitutively in non-negligible quantities lipids and citric acid when glycerol is utilized as the sole substrate, demonstrates a novel regulation of the above-mentioned bioprocesses that never again has been mentioned. On the other hand, growth of both the wild and the mutant strain on crude glycerol at increasing initial glycerol concentrations and constant nitrogen, showed that during non-limiting nitrogen conditions JMY1203 was able to accumulate lipids faster than W29, and the rate of lipid accumulation increased with the glycerol concentration. However, lipid turnover was more significant in the mutant strain once nitrogen was depleted, probably due to deregulation of the remobilization pathway through the induction of triacylglycerol lipases. A strain over-expressing GPD1 has a similar phenotype; GPD1 overexpression in this strain triggered the induction of Tgl3 and Tgl4 lipases (Dulermo and Nicaud, 2011). When lipids enter catabolism via ˇ-oxidation, they are broken down to give acetyl-CoA, which can be reused for citrate production through the TCA cycle (Fig. 1). Qualitative “arrows” of metabolic profiles for JMY1203 and W29 strain under nitrogen limitation are given in Fig. 5. Lipid analysis showed that regardless of the quantity of lipid found inside the cells, neutral lipids (including triacylglycerols) were the main fraction detected (see Table 5). The P fraction was less saturated (as regards principally the FA acid 9,12 C18:2) than N and G + S fractions, consistent with the results reported for several wild-type and genetically engineered strains (André et al., 2009; Papanikolaou and Aggelis, 2009). The FA compositions of cellular lipids produced by Y. lipolytica strains differed according to the

Fig. 5. Arrows indicating, qualitatively, the carbon flow towards biomass, lipid and citric acid production by Yarrowia lipolytica strains W29 and JMY1203.

culture time and the initial concentration of glycerol in the culture medium. These differences seem to be strain dependent; it appears that increasing the concentration of glycerol (or glucose) in the culture medium does not have a systematic common effect on the modification of cellular FAs in the cells of Y. lipolytica (Chatzifragkou et al., 2011; Papanikolaou and Aggelis, 2009; Papanikolaou et al., 2006, 2008, 2009). Crude glycerol is a potential feedstock for biotechnological applications, and is increasingly attracting interest (Metsoviti et al., 2012, 2013; Papanikolaou et al., 2000; Papanikolaou and Aggelis, 2009). There have been a growing number of publications reporting the conversion of crude glycerol into citric acid by wild-type or mutant Y. lipolytica strains. Citmax production of 57.7 g/L (with isocitric acid making up ∼5%, w/w, of the Cit produced) by the strain JMY1203, with a YCit/Glol yield >0.90 g/g, compares favorably with results described for shake-flask experiments for both the absolute (g/L) and relative (g per g of glycerol consumed) production of citric acid. Nevertheless, higher citric acid levels have been reported for conversion in fed-batch bioreactors. A summary of findings for the conversion of crude glycerol to citric acid by Y. lipolytica strains in various fermentation configurations, including the current study is given in Table 7. Nevertheless, even by comparison with bioreactor experiments, the yield of citric acid per unit of glycerol consumed with strain JMY1203 is, to our knowledge, amongst the highest ever reported. As a conclusion, deletion of the PHD1 gene halting the 2methylcitrate pathway at the 2-methylcitrate production level in Y. lipolytica resulted in significant shift of the metabolism under both carbon- and nitrogen-limited conditions when waste glycerol was utilized as carbon and energy source; compared with the performances of the wild strain, the genetically engineered strain produced significantly lower biomass production, and drastically higher ones of cellular lipids (in terms of fat produced per unit of dry weight) and, principally, of citric acid. Under carbon-limited conditions the mutant strain constitutively accumulated lipid and produced citric acid. Total citric acid of 57.7 g/L containing ∼5% w/w of iso-citric acid was produced, with an exceptional conversion yield of total citric acid per glycerol consumed of 0.91 g/g. Y. lipolytica JMY1203 is, therefore, a perfect candidate for glycerolbased biorefineries processes.

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Table 7 Overview of published data for citric acid production from glycerol-based media by Yarrowia lipolytica strains. Strain

Citric acid (g/L)

Glycerol type

Yield (g/g)

Fermentation type

Reference

ACA-DC 50109 Wratislavia 1.31 Wratislavia AWG7 Wratislavia K1 NRRL YB-423 NCIM 3589 ACA-DC 50109 A-101-1.22 ACA-YC 5033 A-101 – Wratislavia K1 – Wratislavia 1.31 Wratislavia AWG7 Wratislavia 1.31 Wratislavia AWG7 N15 – Wratislavia AWG7 – NG40/UV7 – JMY1203

33.6 124.5 88.1 75.7 21.6 77.4 62.5 112.0 50.1 66.5 66.8 53.3 36.8 126.0 157.5 155.2 154.0 19.08 98.0 86.5 63.3 115.0 112.0 57.7c

Crude – – – Pure Crude – – – Pure Crude Pure Crude – – – – Pure – – – – Crude Crude

0.44 0.62 0.46 0.40 0.55 n. r. 0.56 0.60 0.44 0.44 0.43 0.34 0.25 0.63 0.58 0.55 0.78 0.55 0.70 0.59 0.67 0.64 0.90 0.92c

Shake flasks Batch bioreactor – – Shake flasks – Shake flasks Batch bioreactor Shake flasks Batch bioreactor – – – Fed-batch bioreactor – – Repeated batch Shake flasks Fed-batch bioreactor Continuous bioreactora Continuous bioreactorb Fed-batch bioreactor – Shake flasks

Papanikolaou et al. (2002a) Rymowicz et al. (2006) – – Levinson et al. (2007) Imandi et al. (2007) Papanikolaou et al. (2008) Rymowicz et al. (2010) André et al. (2009) ´ Rywinska et al. (2010a) – – – ´ Rywinska et al. (2010b) – – ´ Rywinska and Rymowicz (2010) Kamzolova et al. (2011) – ´ Rywinska et al. (2011) – Morgunov et al. (2013) – Present study

n. r.: Not reported in the paper a D = 0.009 h−1. b D = 0.021 h−1. c Value for total citric acid, iso-citric acid content being ∼5% w/w, of total citric acid.

Acknowledgements The current investigation has been funded by the project entitled “Research on oleaginous microorganisms and development of new biotechnological processes” (Acronym: “MicroOil”), action “ARISTEIA”, in the operational program “Education and Lifelong learning”, 2007-2013 (financial support: GSRT and European Union). We thank Alex Edelman and Associates for excellent help with correcting the English version of the manuscript. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec. 2013.10.025. References Anastassiadis, S., Aivasidis, A., Wandrey, C., 2002. Citric acid production by Candida strains under intracellular nitrogen limitation. Appl. Microbiol. Biotechnol. 60, 81–87. André, A., Chatzifragkou, A., Diamantopoulou, P., Sarris, D., Philippoussis, A., Galiotou-Panayotou, M., Komaitis, M., Papanikolaou, S., 2009. Biotechnological conversions of bio-diesel-derived crude glycerol by Yarrowia lipolytica strains. Eng. Life Sci. 9, 468–478. Athenstaedt, K., Jolivet, P., Boulard, C., Zivy, M., Negroni, L., Nicaud, J.M., Chardot, T., 2006. Lipid particle composition of the yeast Yarrowia lipolytica depends on the carbon source. Proteomics 6, 1450–1459. Barth, G., Gaillardin, C., 1996. Yarrowia lipolytica. In: Wolf, K. (Ed.), Nonconventional Yeasts in Biotechnology, 1. Springer-Verlag, Berlin, Heidelberg, New York, pp. 313–388. Beopoulos, A., Mrozova, Z., Thevenieau, F., Le Dall, M.T., Hapala, I., Papanikolaou, S., Chardot, T., Nicaud, J.M., 2008. Control of lipid accumulation in the yeast Yarrowia lipolytica. Appl. Environ. Microbiol. 74, 7779–7789. Beopoulos, A., Cescut, J., Haddouche, R., Uribelarrea, J.L., Molina-Jouve, C., Nicaud, J.M., 2009a. Yarrowia lipolytica as a model for bio-oil production. Prog. Lipid Res. 48, 375–387. Beopoulos, A., Chardot, T., Nicaud, J.M., 2009b. Yarrowia lipolytica: a model and a tool to understand the mechanisms implicated in lipid accumulation. Biochimie 91, 692–696. Beopoulos, A., Haddouche, R., Kabran, P., Dulermo, T., Chardot, T., Nicaud, J.M., 2012. Identification and characterization of DGA2, an acyltransferase of the DGAT1 acyl-CoA:diacylglycerol acyltransferase family in the oleaginous yeast Yarrowia

lipolytica. New insights into the storage lipid metabolism of oleaginous yeasts. Appl. Microbiol. Biotechnol. 93, 1523–1537. Cescut, J., Accumulation d’acylglycérols par des espèces levuriennes à usage carburant aéronautique: physiologie et performances de procédés. Université de Toulouse, Toulouse, 2009, p. 283. Chatzifragkou, A., Makri, A., Belka, A., Bellou, S., Mavrou, M., Mastoridou, M., Mystrioti, P., Onjaro, G., Aggelis, G., Papanikolaou, S., 2011. Biotechnological conversions of biodiesel derived waste glycerol by yeast and fungal species. Energy 36, 1097–1108. Cheema-Dhaldi, S., Leznoff, C., Halperin, M., 1975. Effect of 2-methylcitrate on citrate metabolism: implications for the management of patients with propionic acidemia and methylmalonic aciduria. Pediat. Res. 9, 905–908. Dulermo, T., Nicaud, J.M., 2011. Involvement of the G3P shuttle and beta-oxidation pathway in the control of TAG synthesis and lipid accumulation in Yarrowia lipolytica. Metab. Eng. 13, 482–491. Fakas, S., Papanikolaou, S., Galiotou-Panayotou, M., Komaitis, M., Aggelis, G., 2006. Lipids of Cunninghamella echinulata with emphasis to gamma-linolenic acid distribution among lipid classes. Appl. Microbiol. Biotechnol. 73, 676–683. Fickers, P., Benetti, P.H., Wache, Y., Marty, A., Mauersberger, S., Smit, M.S., Nicaud, J.M., 2005. Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications. FEMS Yeast Res. 5, 527–543. Fickers, P., Le Dall, M.T., Gaillardin, C., Thonart, P., Nicaud, J.M., 2003. New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J. Microbiol. Methods 55, 727–737. Fontanille, P., Kumar, V., Christophe, G., Nouaille, R., Larroche, C., 2012. Bioconversion of volatile fatty acids into lipids by the oleaginous yeast Yarrowia lipolytica. Bioresour. Technol. 114, 443–449. Gerike, U., Hough, D.W., Russell, N.J., Dyall-Smith, M.L., Danson, M.J., 1998. Citrate synthase and 2-methylcitrate synthase: structural, functional and evolutionary relationships. Microbiol 144, 929–935. Guo, X., Ota, Y., 2000. Incorporation of eicosapentaenoic and docosahexaenoic acids by a yeast (FO726A). J. Appl. Microbiol. 89, 107–115. Imandi, S.B., Bandaru, V.V., Somalanka, S.R., Garapati, H.R., 2007. Optimization of medium constituents for the production of citric acid from byproduct glycerol using Doehlert experimental design. Enzyme Microb. Technol. 40, 1367–1372. Kamzolova, S.V., Fatykhova, A.R., Dedyukhina, E.G., Anastassiadis, S.G., Golovchenko, N.P., Morgunov, I.G., 2011. Citric acid production by yeast grown on glycerolcontaining waste from biodiesel industry. Food Technol. Biotechnol. 49, 65–74. Levinson, W.E., Kurtzman, C.P., Kuo, T.M., 2007. Characterization of Yarrowia lipolytica and related species for citric acid production from glycerol. Enzyme Microb. Technol. 41, 292–295. Liu, X.-Y., Chi, Z., Liu, G.-L., Madzak, C., Chi, Z.-M., 2013. Both decrease in ACL1 gene expression and increase in ICL1 gene expression in marine-derived yeast Yarrowia lipolytica expressing INU1 gene enhance citric acid production from inulin. Mar. Biotechnol. 15, 26–36. Makri, A., Fakas, S., Aggelis, G., 2010. Metabolic activities of biotechnological interest in Yarrowia lipolytica grown on glycerol in repeated batch cultures. Bioresour. Technol. 101, 2351–2358.

314

S. Papanikolaou et al. / Journal of Biotechnology 168 (2013) 303–314

Metsoviti, M., Paramithiotis, S., Drosinos, E.H., Galiotou-Panayotou, M., Nychas, G.J.E., Zeng, A.P., Papanikolaou, S., 2012. Screening of bacterial strains capable of converting biodiesel-derived raw glycerol into 1,3-propanediol, 2,3-butanediol and ethanol. Eng. Life Sci. 12, 57–68. Metsoviti, M., Zeng, A.P., Koutinas, A.A., Papanikolaou, S., 2013. Enhanced 1,3propanediol production by a newly isolated Citrobacter freundii strain cultivated on biodiesel-derived waste glycerol through sterile and non-sterile bioprocesses. J. Biotechnol. 163, 408–418. Mliˇcková, K., Luo, Y., d’Andrea, S., Peˇc, P., Chardot, T., Nicaud, J.M., 2004a. Acyl-CoA oxidase, a key step for lipid accumulation in the yeast Yarrowia lipolytica. J. Mol. Catal. B: Enzymatic 28, 81–85. Mliˇcková, K., Roux, E., Athenstaedt, K., d’Andrea, S., Daum, G., Chardot, T., Nicaud, J.M., 2004b. Lipid accumulation, lipid body formation, and acyl-coenzyme A oxidases of the yeast Yarrowia lipolytica. Appl. Environ. Microbiol. 70, 3918–3924. Morgunov, I.G., Kamzolova, S.V., Lunina, J.N., 2013. The citric acid production from raw glycerol by Yarrowia lipolytica yeast and its regulation. Appl. Microbiol. Biotechnol. 97, 7387–7397. Papanikolaou, S., Aggelis, G., 2002. Lipid production by Yarrowia lipolytica growing on industrial glycerol in a single-stage continuous culture. Bioresour. Technol. 82, 43–49. Papanikolaou, S., Aggelis, G., 2009. Biotechnological valorization of biodiesel derived glycerol waste through production of single cell oil and citric acid by Yarrowia lipolytica. Lipid Technol. 21, 83–87. Papanikolaou, S., Aggelis, G., 2011a. Lipids of oleaginous yeasts. Part I: Biochemistry of single cell oil production. Eur. J. Lipid Sci. Technol. 113, 1031–1051. Papanikolaou, S., Aggelis, G., 2011b. Lipids of oleaginous yeasts. Part II: Technology and potential applications. Eur. J. Lipid Sci. Technol. 113, 1052–1073. Papanikolaou, S., Ruiz-Sanchez, P., Pariset, B., Blanchard, F., Fick, M., 2000. High production of 1,3-propanediol from industrial glycerol by a newly isolated Clostridium butyricum strain. J. Biotechnol. 77, 191–208. Papanikolaou, S., Muniglia, L., Chevalot, I., Aggelis, G., Marc, I., 2002a. Yarrowia lipolytica as a potential producer of citric acid from raw glycerol. J. Appl. Microbiol. 92, 737–744. Papanikolaou, S., Chevalot, I., Komaitis, M., Marc, I., Aggelis, G., 2002b. Single cell oil production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures. Appl. Microbiol. Biotechnol. 58, 308–312. Papanikolaou, S., Galiotou-Panayotou, M., Chevalot, I., Komaitis, M., Marc, I., Aggelis, G., 2006. Influence of glucose and saturated free-fatty acid mixtures on citric acid and lipid production by Yarrowia lipolytica. Curr. Microbiol. 52, 134–142. Papanikolaou, S., Fakas, S., Fick, M., Chevalot, I., Galiotou-Panayotou, M., Komaitis, M., Marc, I., Aggelis, G., 2008. Biotechnological valorisation of raw glycerol discharged after bio-diesel (fatty acid methyl-esters) manufacturing process: production of 1,3-propanediol, citric acid and single cell oil. Biomass Bioeng. 32, 60–71. Papanikolaou, S., Chatzifragkou, A., Fakas, S., Galiotou-Panayotou, M., Komaitis, M., Nicaud, J.M., Aggelis, G., 2009. Biosynthesis of lipids and organic acids by Yarrowia lipolytica strains cultivated on glucose. Eur. J. Lipid Sci. Technol. 111, 1221–1232. Ratledge, C., 1994. Yeasts, moulds, algae and bacteria as sources of lipids. In: Kamel, B.S., Kakuda, Y. (Eds.), Technological Advances in Improved and Alternative Sources of Lipids. Blackie Academic & Professional, London, pp. 235–291.

Ratledge, C., Wynn, J.P., 2002. The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv. Appl. Microbiol. 51, 1–51. ´ Rymowicz, W., Rywinska, A., Zarowska, B., Juszczyk, P., 2006. Citric acid production from raw glycerol by acetate mutants of Yarrowia lipolytica. Chem. Pap. 60, 391–394. ´ Rymowicz, W., Rywinska, A., Marcinkiewicz, M., 2009. High-yield production of erythritol from raw glycerol in fed-batch cultures of Yarrowia lipolytica. Biotechnol. Lett. 31, 377–380. Rymowicz, W., Fatykhova, A.R., Kamzolova, S.V., Rywinska, A., Morgunov, I.G., 2010. Citric acid production from glycerol-containing waste of biodiesel industry by Yarrowia lipolytica in batch, repeated batch, and cell recycle regimes. Appl. Microbiol. Biotechnol. 87, 971–979. ´ ´ Rywinska, A., Rymowicz, W., Zarowska, B., Skrzypinski, A., 2010a. Comparison of citric acid production from glycerol and glucose by different strains of Yarrowia lipolytica. World J. Microbiol. Biotechnol. 26, 1217–1224. ´ Rywinska, A., Rymowicz, W., Marcinkiewicz, M., 2010b. Valorization of raw glycerol for citric acid production by Yarrowia lipolytica yeast. Electron. J. Biotechnol. 13 (4). ´ Rywinska, A., Juszczyk, P., Wojtatowicz, M., Rymowicz, W., 2011. Chemostat study of citric acid production from glycerol by Yarrowia lipolytica. J. Biotechnol. 152, 54–57. ´ Rywinska, A., Juszczyk, P., Wojtatowicz, M., Robak, M., Lazar, Z., Tomaszewska, L., Rymowicz, W., 2013. Glycerol as a promising substrate for Yarrowia lipolytica biotechnological applications. Biomass Bioenergy 48, 148–166. ´ Rywinska, A., Rymowicz, W., 2010. High-yield production of citric acid by Yarrowia lipolytica on glycerol in repeated-batch bioreactors. J. Ind. Microbiol. Biotechnol. 37, 431–435. Sambrook, J., Maniatis, T., Fritsch, E.F., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Tai, M., Stephanopoulos, G., 2013. Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production. Metab. Eng. 15, 1–9. Tsigie, Y.A., Wang, C.-Y., Truong, C.-T., Ju, Y.-H., 2011. Lipid production from Yarrowia lipolytica Po1g grown in sugarcane bagasse hydrolysate. Bioresour. Technol. 102, 9216–9222. Tsigie, Y.A., Wang, C.-Y., Kasim, N.S., Diem, Q.D., Huynh, L.H., Ho, Q.P., Truong, C.T., Ju, Y.-H., 2012. Oil production from Yarrowia lipolytica Po1 g using rice bran hydrolysate. J. Biomed. Biotechnol. 2012, 378384. Uchiyama, H., Ando, M., Toyonaka, Y., Tabuchi, T., 1982. Subcellular localization of the methylcitric-acid-cycle enzymes in propionate metabolism of Yarrowia lipolytica. Eur. J. Biochem. 125, 523–527. Uchiyama, H., Tabuchi, T., 1976. Properties of methylcitrate synthase from Candida lipolytica. Agric. Biol. Chem. 40, 1411–1418. Upton, A.M., McKinney, J.D., 2007. Role of the methylcitrate cycle in propionate metabolism and detoxification in Mycobacterium smegmatis. Microbiology 153, 3973–3982. Wang, Z.P., Xu, H.M., Wang, G.Y., Chi, Z., Chi, Z.M., 2013. Disruption of the MIG1 gene enhances lipid biosynthesis in the oleaginous yeast Yarrowia lipolytica ACA-DC 50109. Biochim. Biophys. Acta 1831, 675–682.