RESEARCH ARTICLE

A comparative study on glycerol metabolism to erythritol and citric acid in Yarrowia lipolytica yeast cells  ska Ludwika Tomaszewska, Magdalena Rakicka, Waldemar Rymowicz & Anita Rywin Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Wrocław, Poland

ska, Correspondence: Anita Rywin Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, skiego Str. 37/41, 51-630 Wrocław, Chełmon Poland. Tel.: +48 713207736; fax: +48 713207794; e-mail: [email protected] Received 26 March 2014; revised 19 June 2014; accepted 8 July 2014. Final version published online 04 August 2014. DOI: 10.1111/1567-1364.12184 Editor: Guenther Daum Keywords Yarrowia lipolytica; glycerol; erythritol; citric acid; transketolase; erythrose reductase.

Abstract Citric acid and erythritol biosynthesis from pure and crude glycerol by three acetate-negative mutants of Yarrowia lipolytica yeast was investigated in batch cultures in a wide pH range (3.0–6.5). Citric acid biosynthesis was the most effective at pH 5.0–5.5 in the case of Wratislavia 1.31 and Wratislavia AWG7. With a decreasing pH value, the direction of biosynthesis changed into erythritol synthesis accompanied by low production of citric acid. Pathways of glycerol conversion into erythritol and citric acid were investigated in Wratislavia K1 cells. Enzymatic activity was compared in cultures run at pH 3.0 and 4.5, that is, under conditions promoting the production of erythritol and citric acid, respectively. The effect of pH value (3.0 and 4.5) and NaCl presence on the extracellular production and intracellular accumulation of citric acid and erythritol was compared as well. Low pH and NaCl resulted in diminished activity of glycerol kinase, whereas such conditions stimulated the activity of glycerol-3-phosphate dehydrogenase. The presence of NaCl strongly influenced enzymes activity – the effective erythritol production was correlated with a high activity of transketolase and erythrose reductase. Therefore, presented results confirmed that transketolase and erythrose reductase are involved in the overproduction of erythritol in the cells of Y. lipolytica yeast.

YEAST RESEARCH

Introduction Polyols, including erythritol, glycerol, ribitol, and arabitol, might be formed as an integral part of normal growth processes, however, in different proportions, depending on the strain and growth conditions (R€ ohr et al., 1983; Omar et al., 1992; Lucca et al., 2002). In fungi, the presence of sugar alcohols, synthesized as byproducts during citric acid fermentation by A. niger, was first described by R€ ohr in 1983 (R€ ohr et al., 1983). Today, it is known that citric acid fungi may produce sugar alcohols during biosynthesis – mainly glycerol followed by erythritol, arabitol, and mannitol (Dijkema et al., 1985; Omar et al., 1992). With respect to yeasts, knowledge in this field is scarce and a few studies only describe the formation of polyols during the biosynthesis of citric acid. The biosynthesis of citric acid from n-paraffins and glucose by Candida lipolytica has been shown to result in small amounts of mannitol, arabitol, and erythritol produced (Hattori & Suzuki, 1974b; Anastassiadis et al., 2002). However, the mechanisms of those compounds formation have not ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

been elucidated. Anastassiadis et al. (2002) reported on the possibility of accumulation and then utilization of sugar alcohols during biosynthesis of citric acid from glucose by yeast, but types and concentrations of the compounds were not specified. In the last few decades, several studies have been described that addressed the production of citric acid from glycerol by the yeast Yarrowia lipolytica (Papanikolaou et al., 2002; Rywi nska et al., 2013). It was identified that during citric acid biosynthesis from glycerol, erythritol and mannitol were present as the predominant byproducts in the culture broths (Rywi nska et al., 2010). The capability of overproducing erythritol was observed for osmophilic yeast from the following genera: Pichia, Zygopichia, Candida, Debaryomyces, Moniliella, Torula, Torulopsis, Trigonopsis, Trichosporon, Trichosporonoides, Pseudozyma, and Ustilago (Hajny et al., 1964; Jeya et al., 2009; Moon et al., 2010), some fungi such as Penicillium (Lee & Lim, 2003) and for bacteria, for example, Leuconostoc oenos (Veiga-Da-Cunha et al., 1992). Erythritol biosynthesis was reported to be strongly dependent on, inter alia, changes in osmotic pressure, pH FEMS Yeast Res 14 (2014) 966–976

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Glycerol metabolism to erythritol

and temperature of medium, type of substrate and its concentration, sources of nitrogen and phosphorus, and additional factors such as chloride, copper, and manganese ions (Kim et al., 1997; Lee et al., 2000; Lin et al., 2001; Jeya et al., 2009; Savergave et al., 2011). Worthy of notice is that, until today, glucose, fructose, sucrose, and starch hydrolysates have been used as the major substrates for erythritol production by osmophilic yeast in research performed on a laboratory as well as industrial scale (Moon et al., 2010). Unconventional substrates, such as glycerol, have been reported as not suitable for erythritol biosynthesis (Jeya et al., 2009). In the presented work, we have compared the capability of three acetate-negative mutants of Y. lipolytica for extraand intracellular production of citric acid and erythritol from glycerol. The experiments were performed under conditions promoting biosynthesis of these metabolites. The main objective of this study was to identify the impact of the factors responsible for overproduction of erythritol from glycerol in cells of Y. lipolytica yeast and their effect on the activity of enzymes involved in the process.

Materials and methods Wratislavia 1.31, Wratislavia AWG7, and Wratislavia K1 strains of Y. lipolytica used in this study were from the yeast culture collection belonging to the Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences in Poland. They are acetate-negative mutants incapable of growth on acetate as the sole carbon and energy source. The strains have been frequently used in the studies on citric acid and erythritol biosynthesis since 1996. The mutants of Y. lipolytica (Wratislavia 1.31 and Wratislavia AWG7) were isolated after exposure to UV irradiation. Y. lipolytica Wratislavia K1 strain was isolated from Wratislavia 1.31 in the course of continuous citric acid production from glucose in nitrogen-limited chemostat at a dilution rate of 0.016 h 1. The history of the Y. lipolytica strains used in this work was described by Rywi nska et al. (2012). The growth medium for inoculum was prepared as described by Tomaszewska et al. (2012). An inoculum of 0.2 L was introduced into a bioreactor containing 1.8 L of production medium which consisted of: 150 g of glycerol, 3 g of NH4Cl, 1 g of MgSO4x7H2O, 0.2 g of KH2PO4, and 1 g of yeast extract per liter of tap water. Pure glycerol or crude glycerol from methyl ester production (SG BODDINS GmbH; Germany) (containing 86% wt/wt of glycerol, 6.5% wt/wt of NaCl and methanol – below 0.2%) was used as carbon and energy sources in the media. To obtain 150 g L 1 of glycerol in the FEMS Yeast Res 14 (2014) 966–976

production medium, 175 g L 1 of the crude glycerol was added. Additionally, 32.5 g L 1 NaCl in the production medium with crude glycerol was obtained by adding 21 g L 1 of NaCl. The cultivations were carried out in a 5-L stirred-tank reactor (Biostat B Plus, Sartorius; Germany) with a working volume of 2 L at 30 °C, and the aeration rate was fixed at 0.36 vvm. The stirrer speed was set at 800 rev min 1. Different values of pH: 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.5 were maintained automatically by the addition of 20% or 40% (w/v) NaOH solution. All the cultures were cultivated till the glycerol had been completely consumed. Samples were withdrawn 2–3 times per day. All experiments were performed in three replicates, and the results are presented as mean values. Analytical methods

For biomass determination, samples were centrifuged and washed two times with distilled water, and cells were harvested by filtration on 0.45 lm pore-size membranes and dried to a constant weight at 105 °C. In the supernatant glycerol, citric acids, erythritol, mannitol, and arabitol were determined with the use of HPLC (Tomaszewska et al., 2012). Isocitric acid was determined using an enzymatic method as described by Goldberg & Ellis (1983). Intracellular metabolites were analyzed in biomass obtained at the end of the cultures as described before (Tomaszewska et al., 2012). The osmotic pressure was measured with the use of Marcel OS 3000 Osmometer (Marcel, Poland). Enzyme assays

The enzyme activities were measured during the growth period and at the end of production phase. The samples of 0.2 L were withdrawn from the bioreactors, centrifuged (10 min, 4 °C, 4300 g), and washed twice with a 0.1 M phosphate buffer (pH 7.4). Next, the biomass was resuspended in 0.05 L of a disruption buffer (1 mM EDTA, 5 mM PMSF, 5 mM DTT, 0.1 M phosphate buffer pH 7.4), disrupted with glass beads using Sonics VCX500 (30 min, 4 °C), and centrifuged (20 min, 4 °C, 9800 g). Activity of the following enzymes was determined in the supernatant: glycerol kinase, GK, (EC 2.7.1.30) and citrate synthase, CS, (EC 2.3.3.1) (Kamzolova et al., 2008), glycerol-3-phosphate dehydrogenase, GPDH, (EC 1.1.1.8) (White & Kaplan, 1969), transketolase, TK, (EC 2.2.1.1) (Sugimoto & Shiio, 1989), and erythrose reductase, ER, (EC 1.1.1.21) (Lee et al., 2003). Protein content was determined by the Lowry’s method. One unit (U) of the enzyme activity was defined as 1 lmol of NADH/NADPH consumed or produced per 1 min (k = 340 nm). The ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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enzyme activities were expressed as U per mg of protein (U mg 1 of protein) or U per mg of yeast biomass dry weight (U mg 1 of DW).

Results Influence of pH on citric acid and sugar alcohols biosynthesis by acetate-negative mutants of Y. lipolytica

The effect of pH (from 3.0 to 6.5) on biomass, citric acid, and sugar alcohols production by three acetate-negative mutants of Y. lipolytica – Wratislavia 1.31, Wratislavia AWG7, and Wratislavia K1 – was investigated in batch cultures (Table 1). The final biomass concentration reached a comparable level and was from 16.9 to 22.0 g L 1 for Wratislavia 1.31 and Wratislavia K1. The concentration of biomass in the cultures with Wratislavia AWG7 strain was slightly lower and varied from 15.5 to 18.0 g L 1. As shown in Table 1, at low pH (3.0), the production of citric acid did not exceed 2.6 g L 1. Citric acid was produced more effectively when pH value ranged between 3.5 and 6.5. The highest acid concentration reached 85.7 g L 1 and was obtained for Wratislavia AWG7 strain at pH 5.5, whereas 76.0 g L 1 was identified at pH 5.0 for Wratislavia 1.31 strain. The best citric acid production yield, 0.52 g g 1, was observed for Wratislavia AWG7 strain at pH 5.5. The highest value of citric acid volumetric production rate, of about 1.1 g L 1 h 1, was obtained at pH 5.5 for both Wratislavia 1.31 and Wratislavia AWG7. The specific rate of acid production was also the highest at pH 5.5 and reached 0.065 and 0.062 g g 1 h 1 for Wratislavia AWG7 and Wratislavia 1.31, respectively. Extracellular erythritol, mannitol, and arabitol levels decreased with an increasing pH value. However, irrespective of medium pH erythritol was produced as the predominant sugar alcohol, whereas arabitol synthesis was the lowest. The highest concentration of erythritol, 40.7 g L 1, was produced by Wratislavia K1 strain at pH 3.0. For more in-depth investigations on the influence of pH, the intracellular accumulation of citric acid and sugar alcohols was determined at pH 3.0 and 5.5 (Table 2). The highest contents of erythritol were accumulated in the cells of Wratislavia K1 strain and reached 72.0 and 89.9 mg g 1 of DW at pH 3.0 and 5.5, respectively. In comparison with Wratislavia 1.31 and Wratislavia AWG7, lower concentration of mannitol was observed in the cells of Wratislavia K1. The highest concentrations of intracellular citric acid were found at pH 5.5 for Wratislavia AWG7 and Wratislavia 1.31. For the examined strains, the ratio of intra- to extracellular erythritol concentrations was higher at pH 5.5 than at pH 3.0. In turn, the ratio of intra- to extracellular citric acid was higher at low pH. ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

L. Tomaszewska et al.

Influence of NaCl on sugar alcohols and citric acid formation by Y. lipolytica Wratislavia K1 strain

The effect of NaCl addition on erythritol and mannitol production from pure and crude glycerol was investigated at pH 3.0. For comparison, the same experiment was carried out at pH 4.5, where additionally, production of citric acid was taken into account. As shown in Table 3, the parameters of erythritol production such as yield and productivity were higher in the medium with pure glycerol and NaCl (3.25%), than in the culture without salt. Moreover, erythritol biosynthesis was strongly affected by the use of crude glycerol and crude glycerol supplemented with NaCl. The highest yield of erythritol production (0.40 g g 1) was achieved at pH 3.0 in the medium with crude glycerol and NaCl, while the best value of volumetric erythritol production rate in the culture with pure glycerol supplemented with NaCl. In the culture run at pH 4.5 with pure glycerol, the addition of salt enhanced erythritol biosynthesis but had a negative effect on citric acid production. At higher pH, in the cultures with crude glycerol, the concentrations of erythritol and citric acid were comparable. The presence of NaCl in the medium at pH 3.0, as well as the use of crude glycerol, strongly affected the intracellular amounts of erythritol and mannitol (Table 4). With addition of NaCl, the concentration of erythritol increased more than three times in comparison with the culture with pure glycerol without salt. On the contrary, the concentration of mannitol decreased and did not exceed 4 mg g 1 of DW when salt was added to the pure-glycerol medium. At pH 4.5, the cells accumulated from 20.5 to 57.2 mg g 1 of citric acid and from 100.6 to 204.1 mg g 1 of erythritol. As also described earlier, at lower pH, the concentration of erythritol and mannitol was significantly dependent on the presence of salt in the culture medium. At pH 3.0 and pH 4.5, the ratio of intra- to extracellular erythritol concentration was similar in the cultures with NaCl and with crude glycerol. It is interesting to note that despite the difference in the content of mannitol in the cells derived from the culture without and with salt addition, the ratio of intra- to extracellular mannitol levels was found to be similar, irrespective of salt presence; however, at pH 3.0, the values were significantly lower than at pH 4.5. The effect of pH and NaCl on enzyme activities during biosynthesis processes by Y. lipolytica Wratislavia K1

The activity of selected enzymes responsible for glycerol conversion to erythritol and citric acid (Fig. 1) was FEMS Yeast Res 14 (2014) 966–976

FEMS Yeast Res 14 (2014) 966–976

Biomass (g L 1)

Erythritol (g L 1)

nd, not determined.

Y. lipolytica Wratislavia 1.31 3.0 19.0  2.1 26.2  2.8 3.5 19.0  2.2 18.0  2.2 4.0 21.8  1.8 13.0  2.1 5.0 19.0  2.1 9.0  2.1 5.5 18.0  1.4 5.0  1.6 6.5 22.0  2.1 1.8  0.3 Y. lipolytica Wratislavia AWG7 3.0 16.4  0.6 25.7  1.3 3.5 16.2  0.8 25.0  2.0 4.0 18.0  1.2 12.0  1.0 5.0 17.4  1.6 11.0  1.0 5.5 16.4  1.4 10.0  1.2 6.5 15.5  0.7 1.5  0.3 Y. lipolytica Wratislavia K1 3.0 19.3  1.5 40.7  2.6 3.5 18.9  1.7 31.5  2.5 4.0 18.5  0.7 29.0  1.6 5.0 16.9  1.5 27.5  2.1 5.5 17.3  1.7 26.5  2.5 6.5 18.8  1.3 11.8  2.2

pH 1.2 1.4 1.2 0.8 0.8 0.6 1.2 2.0 1.2 0.8 0.6 0.3 0.9 1.2 1.1 0.6 0.7 0.2

                 

16.8 15 11 7.2 8.2 6.0 17.1 14.2 8.2 6.5 4.5 3.5 15.1 5.3 6.8 4.4 6.3 1.1

Mannitol (g L 1)

2.9 2.3 1.6 0.4 0.2 0.4

2.7 2.4 1.9 ≤ 0.1 ≤ 0.1 1.7

3.7 1.5 1.7 ≤ 0.1 ≤ 0.1 1.7

      0.9 0.7 0.4 0.2 0.2 0.2

 0.3

 0.5  0.6  0.3

 0.2

 0.7  0.2  0.2

Arabitol (g L 1)

2.6 13.2 41.7 63.7 65.0 64.9

0.9 8.8 36.9 69.7 85.7 75.6

2.6 25.0 45.5 76.0 73.0 58.7

     

     

     

1.1 2.1 3.5 4.9 2.5 2.7

0.2 0.7 1.4 2.8 4.1 6.3

0.8 2.1 1.4 4.2 2.8 3.5

Citric acid (g L 1)

nd 0.9 2.3 3.3 4.0 3.2

nd 0.9 4.7 3.3 3.1 4.4

nd 0.9 4.5 4.0 4.5 7.3

    

    

    

0.1 0.1 0.2 0.3 0.3

0.2 0.5 0.4 0.3 0.7

0.2 0.6 0.5 0.6 0.7

Isocitric acid (g L 1)

0.28 0.19 0.19 0.18 0.18 0.07

0.17 0.14 0.08 0.07 0.06 0.01

0.20 0.12 0.08 0.06 0.03 0.01

YERY (g g 1)

0.52 0.43 0.39 0.37 0.29 0.16

0.32 0.31 0.16 0.15 0.12 0.02

0.40 0.22 0.22 0.12 0.08 0.02

QERY (g L 1 h 1) 1

0.027 0.023 0.021 0.022 0.017 0.009

0.020 0.019 0.009 0.008 0.008 0.001

0.021 0.012 0.010 0.006 0.004 0.001

qERY (g g h 1)

– 0.08 0.28 0.41 0.43 0.40

– 0.05 0.26 0.46 0.52 0.50

0.02 0.17 0.28 0.48 0.47 0.38

YCA (g g 1)

– 0.18 0.56 0.85 0.72 0.89

– 0.11 0.48 0.92 1.07 0.97

0.04 0.31 0.76 0.99 1.12 0.79

QCA (g L

1

h 1)

Table 1. Influence of pH value on the production of biomass, citric acids, and sugar alcohols by the acetate-negative mutant strains of Y. lipolytica growing on glycerol media 1

– 0.009 0.030 0.050 0.042 0.048

– 0.006 0.027 0.053 0.065 0.063

0.002 0.016 0.035 0.052 0.062 0.036

qCA (g g

h 1)

Glycerol metabolism to erythritol

969

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L. Tomaszewska et al.

970

Table 2. Intracellular accumulation of sugar alcohols and citric acid in the cells of acetate-negative mutants of Y. lipolytica depending on medium pH when grown on glycerol Erythritol (mg g 1 DW)

Ratio of intra/extra*

Mannitol (mg g 1 DW)

Ratio of intra/extra*

1.31 AWG7 K1

54.9  3.5 21.0  2.2 72.0  6.8

0.040 0.013 0.034

61.5  5.8 55.3  3.3 41.4  1.8

0.070 0.053 0.053

24.7  0.2 9.3  1.1 11.5  1.1

0.170 0.170 0.085

1.31 AWG7 K1

45.6  3.3 31.2  3.1 89.9  6.3

0.164 0.051 0.053

32.6  2.4 23.6  0.4 9.7  0.6

0.072 0.086 0.026

90.7  7.6 105.5  10.0 77.6  7.9

0.022 0.021 0.018

Strain pH 3.0 Wratislavia Wratislavia Wratislavia pH 5.5 Wratislavia Wratislavia Wratislavia

Citric acid (mg g 1 DW)

Ratio of intra/extra*

*The ratio of concentrations of intra to extracellular metabolites was calculated as a result of intracellular metabolite accumulation (mg g 1) per extracellular amount of metabolite (in mg) produced by 1 g of DW.

Table 3. Comparison of parameters of citric acid and erythritol biosynthesis from glycerol by Y. lipolytica Wratislavia K1 strain at pH 3.0 and pH 4.5 depending on NaCl presence

Substrate, NaCl pH 3.0 Pure glycerol Pure glycerol with NaCl* Crude glycerol Crude glycerol with NaCl* pH 4.5 Pure glycerol Pure glycerol with NaCl* Crude glycerol Crude glycerol with NaCl*

Time (h)

Biomass (g L 1)

Erythritol (g L 1)

Mannitol (g L 1)

78 75

19.3  0.5 20.2  1.0

40.7  2.6 64.0  2.6

15.1  0.9 2.5  0.5

76 103

25.0  1.7 20.3  1.3

56.0  4.0 62.0  5.0

4.3  0.4 2.7  0.5

69.5 101

23.7  1.4 17.3  1.3

32.2  1.4 38.7  2.2

6.5  0.8 0.7  0.1

70 138

24.9  1.7 18.9  1.5

35.5  1.9 41.8  2.4

2.7  0.5 1.6  0.4

Arabitol (g L 1)

Citric acid (g L 1)

YERY (g g 1)

QERY (g L 1 h 1)

YCA (g g 1)

QCA (g L

2.6  1.1 0.5  0.1

0.28 0.42

0.52 0.85

– –

– –

0.5  0.1 1.7  0.2

0.37 0.40

0.74 0.60

– –

– –

0.3  0.1 0.2  0.1

45.9  3.9 38.3  1.3

0.22 0.28

0.46 0.38

0.31 0.27

0.66 0.38

0.5  0.1 ≤ 0.1

37.0  2.0 40.6  2.6

0.25 0.26

0.51 0.30

0.26 0.26

0.53 0.29

2.9  0.9 3.6  0.5 ≤ 0.1 ≤ 0.1

1

h 1)

*NaCl in the medium was supplemented to the total concentration of 3.25%. YERY, YCA, yields of erythritol and citric acid production (g of the product per g of utilized glycerol); QERY, QCA, volumetric production rates of erythritol and citric acid.

Table 4. Impact of pH on the intracellular accumulation of sugar alcohols and citric acid in the cells of Y. lipolytica Wratislavia K1 grown at pH 3.0 and 4.5 on glycerol media without and with NaCl

Substrate, NaCl pH 3.0 Pure glycerol Pure glycerol with NaCl† Crude glycerol Crude glycerol with NaCl† pH 4.5 Pure glycerol Pure glycerol with NaCl† Crude glycerol Crude glycerol with NaCl†

Erythritol (mg g 1 DW)

Ratio of intra/extra*

Mannitol (mg g 1 DW)

Ratio of intra/extra*

Citric acid (mg g 1 DW)

Ratio of intra/extra*

72.0 257.2 210.3 212.2

   

6.8 11.2 12.1 12.2

0.034 0.081 0.094 0.070

41.4  4.6 4.0  0.5 ≤ 1.0 7.9  1.0

0.053 0.036 – 0.059

11.5 1.0 5.2 1.8

   

1.1 0.1 0.5 0.1

0.085 0.040 0.260 0.021

100.6 204.1 121.2 190.5

   

7.4 10.8 8.6 10.0

0.062 0.091 0.085 0.086

39.4 11.4 41.3 11.6

   

0.130 0.280 0.380 0.140

57.2 23.4 29.1 20.5

   

7.0 0.3 0.9 0.2

0.029 0.010 0.020 0.010

5.2 0.8 4.0 1.0

*The ratio of concentrations of intra to extracellular metabolites was calculated as a result of intracellular metabolite accumulation (mg g 1) per extracellular amount of metabolite (in mg) produced by 1 g of DW. † NaCl in the medium was supplemented to the total concentration of 3.25%.

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FEMS Yeast Res 14 (2014) 966–976

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Glycerol metabolism to erythritol

Glycerol TCA

1

Pyruvate

Glycerol-3-P 2 Dihydroxyacetone-P

4 Fructose

Glyceraldehyde-3-P

Mannitol 6 Mannitol-1-P

5 3 Fructose-6-P

Fructose-1,6-P

Glucose-6-P

Glucose

7 Arabitol

9

Xylulose

Xylulose-5-P

Erythrose 10

Erythrose-4-P

Erythritol

Sedoheptulose-5-P 7

6-P-gluconolactone

8

9 Glyceraldehyde-3-P Rybose-5-P Rybulose

Rybulose-5-P

Fig. 1. Hypothetical pathways of glycerol conversion into polyhydroxy alcohols in Yarrowia lipolytica yeast. 1 glycerol kinase (GK); 2 NAD+ glycerol-3-P dehydrogenase (GPDH); 3 mannitol dehydrogenase; 4 hexokinase; 5 mannitol-1-P dehydrogenase; 6 mannitol 1-phosphatase; 7 transketolase (TK); 8 transaldolase; 9 arabitol dehydrogenase; 10 erythrose reductase (ER).

determined in cells of Y. lipolytica Wratislavia K1 derived from the cultures with pure-glycerol media, both without and with NaCl addition, conducted at pH 3.0 and 4.5 (Table 5). To compare the changes in the activity throughout the process, the samples for enzyme assays were collected in the growth phase and at the end of the stationary phase of each culture. During the process, the changes of osmotic pressure of culture broth were monitored. The initial osmotic pressure was about 2.0 and

3.0 Osm kg 1 when medium without and with NaCl was applied, respectively (data not presented), and it decreased by about 1 Osm kg 1 at the end of all the cultures. Glycerol kinase (GK) activity was affected by both pH and salt presence. The highest GK activity was observed in the process run at pH 4.5 without NaCl where it reached 0.053 and 0.037 U mg 1 of protein in growth and stationary phase, respectively. In the cultures with

Table 5. Comparison of enzymatic activities and medium osmotic pressure during erythritol biosynthesis from glycerol by Y. lipolytica Wratislavia K1 strain depending on NaCl presence at pH 3.0 and 4.5 Osmotic pressure Substrate, NaCl pH 3.0 Pure glycerol Pure glycerol with NaCl* pH 4.5 Pure glycerol Pure glycerol with NaCl*

GK

GPDH

TK

1

ER

CS

1

1

1

Culture phase

Osm kg

GP SP GP SP

1.6 1.0 2.9 1.9

0.020 0.026 0.026 0.010

3.70 2.92 5.35 1.43

0.022 0.008 0.020 0.018

3.93 0.86 4.18 2.48

0.057 0.009 0.070 0.024

10.37 1.01 14.47 3.25

0.173 0.084 0.230 0.185

31.46 9.51 47.79 25.25

0.784 0.656 0.754 0.792

142.20 74.41 156.38 108.19

GP SP GP SP

1.6 1.0 2.8 2.1

0.053 0.037 0.023 0.018

6.77 2.61 5.55 1.89

0.004 0.005 0.016 0.005

0.45 0.35 3.78 0.56

0.042 0.036 0.064 0.004

5.33 2.49 15.40 0.47

0.115 0.062 0.164 0.124

14.71 4.31 39.44 12.99

0.513 0.419 0.714 0.805

65.51 29.31 171.70 84.17

1

U mg

1

Ug DW

U mg

1

Ug DW

U mg

1

Ug

1

DW

U mg

1

Ug DW

U mg

1

Ug DW

*NaCl in the medium was supplemented to the total concentration of 3.25%. GP, growth phase; SP, stationary phase; GK, glycerol kinase; GPDH, glycerol-3-phosphate dehydrogenase; TK, transketolase; ER, erythrose reductase; CS, citrate synthase.

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salt addition or at lower pH value, the GK activity was significantly lower, which was observed especially in the stationary phase of the processes. The activity of glycerol3-phosphate dehydrogenase (GPDH) was higher at low pH values. In the growth phase of the processes conducted at pH 3.0, the enzyme activity was around 0.020 U mg 1 of protein, whereas it was even 5 times lower at pH 4.5. Furthermore, it was observed that at low pH and NaCl presence, the GPDH activity remained at a comparatively high level in the stationary phase. The highest activity of TK was determined in the growth phase of the cultures conducted at pH 3.0 and in the cultures with salt addition where it ranged from 0.057 to 0.070 U mg 1 of protein. In such conditions, the activity of erythrose reductase (ER) was also noted to be the highest, as it reached 0.230 and 0.185 U mg 1 of protein, respectively, in growth and stationary phase. At higher pH values, the enzyme activity decreased, whereas NaCl presence resulted in the enhanced ER activity. In the cultures with salt, the activity of the enzyme remained at a comparatively high level throughout the process, whereas it decreased by half in the cultures without NaCl. The activity of citrate synthase (CS) was at a comparable level in the cultures conducted at pH 3.0 and in the culture at pH 4.5 with salt addition. When salt was not applied in the culture run at pH 4.5, the CS activity was significantly lower and reached 0.513 and 0.419 U mg 1 of protein in growth and stationary phase, respectively.

Discussion It has already been reported that acetate-negative mutants of Y. lipolytica are capable of producing high amounts of citric acid with relatively low concentration of isocitric acid (Rywi nska et al., 2010). Moreover, it was found that during citric fermentation, polyhydroxy alcohols might be formed in different amounts depending on the strain applied. Because of some differences observed between these strains, a detailed study on the excretion of polyols was necessary. Results presented in this work showed significant differences in citric acid and polyols production abilities of the acetate-negative mutants of Y. lipolytica. At low pH (3.0), the production of citric acid was very low. Only little information regarding citric acid production by yeast at low pH values can be found in the literature. C. lipolytica ATCC 20228 strain, which has been cited in a patent (Nubel et al., 1979), did not require regulation of pH. Kim et al. (1987) reported that when the fermentation broth in the continuous-stirred-tank-membrane-reactor was maintained at pH 2.8, cellular activity declined. According to literature, citrate production by yeast is efficient in nitrogen-limited media with glucose, sucrose, ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

L. Tomaszewska et al.

ethanol, rapeseed-oil, or sunflower-oil used as a carbon source, in which pH is maintained in values ranging from 4.5 to 6.5 (Arzumanov et al., 2000; Anastassiadis et al., 2002; Kamzolova et al., 2005; Anastassiadis & Rehm, 2006; F€ orster et al., 2007; Moeller et al., 2007). The results of the present work showed that pH values of 5.0 and 5.5 were optimal for citric acid fermentation by Y. lipolytica on glycerol-containing media. The best parameters of citric acid production were obtained for Wratislavia AWG7 and Wratislavia 1.31 strains. The comparison of the ratio of intra- to extracellular citric acid concentration suggested that citric acid was more actively excreted by the cells at pH 3.0. While at low pH citric acid concentration was very low, in such conditions the sugar alcohols were produced in significant amounts. Our results showed that acetatenegative mutants of Y. lipolytica produced erythritol and mannitol as the predominant polyols although arabitol was also detected. Irrespective of pH value, the best erythritol-producing strain was found to be Wratislavia K1 which produced from 11.8 g L 1 of the polyol at pH 6.5 to 40.7 g L 1 at pH 3.0. In a similar manner, the glycerol-requiring mutant of Candida zeylanoides produced citric acid from n-paraffins at high pH values, while at lower pH citric acid production was suppressed and erythritol formation was enhanced (Hattori & Suzuki, 1974b). The addition of NaCl (32.5 g L 1) resulted in the increase of erythritol concentration and the simultaneous decrease of extra- and intracellular citrate production, irrespective of pH value. Hattori & Suzuki (1974a) investigated the impact of NaCl, in the range of 0–30 g L 1, on erythritol biosynthesis from n-alkanes by C. zeylanoides KY 6166. The presence of 30 g L 1 of the salt enhanced erythritol production from 26.9 to 38.2 g L 1 and decreased citric acid concentration from 12.2 to 10.1 g L 1. However, Lin et al. (2001) reported that in the process with Moniliella, the increase of NaCl from 29.2 to 58.4 g L 1 resulted in a successive decrease of erythritol amounts. In turn, when yeast from Torula genus was applied, the addition of 58.4 g L 1 of NaCl did not affect the erythritol biosynthesis (Hajny et al., 1964). Generally, the cells of acetate-negative mutants of Y. lipolytica accumulated higher amounts of erythritol and mannitol at pH 3.0 than at pH 5.5. However, the concentration of intracellular erythritol was the highest for Wratislavia K1 cells and comparable at pH 3.0, 4.5, and 5.5. It is interesting that the ratio of intra- to extracellular concentration of erythritol at pH 3.0 and pH 5.5 was also the most similar, which may suggest that the ability to maintain erythritol fluidity through membranes remains unchanged in a wide range of pH values. On the other hand, under the influence of salt in the production FEMS Yeast Res 14 (2014) 966–976

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medium, a drastic increase of intracellular erythritol level in the cells of Wratislavia K1 strain was observed, which indicated that erythritol was not metabolized under osmotic stress. These findings were consistent to the earlier experiment concerning the ability to assimilate various carbon sources and resistance to cycloheximide, performed with the use of commercial API ID 32 C test (Rywi nska et al., 2012). The obtained results indicated disorders in the metabolism of Wratislavia K1, as it was the only strain of Y. lipolytica that did not utilize erythritol. The present experiments and data obtained in our recent investigations (Rymowicz et al., 2009; Tomaszewska et al., 2012) clearly showed that low pH and NaCl addition were one of the key factors enhancing production of polyhydroxy alcohols from glycerol by acetatenegative mutants of Y. lipolytica. Crude glycerol, derived from biodiesel production, contains different contaminations (including inorganic salts) that might be present in the concentration of 6–8%, depending on the catalyst used for biodiesel reaction (Rywi nska et al., 2013). It is worthy of notice that the application of an inexpensive crude substrate might result in better parameters of biosynthesis of different compounds, including erythritol (Tomaszewska et al., 2012; Rywi nska et al., 2013). In this study, the best results were achieved in the cultures with pure substrate supplemented with NaCl as well as with crude glycerol media. To date, biochemical pathways involved in the regulation of erythritol production from glycerol by Y. lipolytica have not been studied in depth. The formation of erythritol and arabitol in osmophilic yeast can be assumed to proceed by combinations and modifications of the Embden–Meyerhof and pentose–phosphate pathways including the action of transketolase (TK) (Spencer & Spencer, 1978). Glycerol may be assimilated via either the phosphorylation or the oxidative pathway (Wang et al., 2001). The results presented by Makri et al. (2010) suggest that Y. lipolytica uses only the phosphorylation pathway. This route of glycerol assimilation includes glycerol phosphorylation by glycerol kinase (GK) to produce glycerol-3-phosphate, which is then converted to dihydroxyacetone phosphate by NAD+-glycerol-3-phosphate dehydrogenase (GPDH) (Fig. 1). According to Ermakova & Morgunov (1987) and Morgunov et al. (2004), some strains of Y. lipolytica also possessed a FAD- glycerol3-phosphate dehydrogenase activity, which catalyzes the dehydratation of glycerophosphate to dihydroxyacetone phosphate. The activities of GK and GPDH enzymes during erythritol biosynthesis from glycerol by Y. lipolytica Wratislavia K1 varied in dependence of pH values and NaCl presence in the medium. The highest activity of GK was determined in the yeast cells derived from the culture conducted at pH 4.5 without salt addition. In FEMS Yeast Res 14 (2014) 966–976

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other culture variants, the enzyme activity was about two times lower, which demonstrated the inhibitory effect of low pH and salt presence on GK activity. The significant decrease of GK activity after salt addition was observed also in the cells of Z. rouxii NRRL Y-998 and S. cerevisiae H44-3D (van Zyl et al., 1991; Albertyn et al., 1994). In the cells of Debaryomyces hansenii (Zopf) van Rij 26, the 80 g L 1 NaCl addition resulted in the decrease of GK activity from 0.026 to 0.018 U mg 1 of protein (Adler et al., 1985). Moreover, when comparing GK activity in logarithmic and stationary phases, it was observed that the enzyme activity decreased throughout the process, which is in accordance with our results. In this study, comparatively low activity of GK at the end of erythritol biosynthesis process was probably due to low concentration of glycerol but possibly also due to a high level of erythritol, which was reported to have an inhibitory effect on GK in Candida mycoderma (Eisenthal et al., 1974) and Hansenula polymorpha (de Koning et al., 1987). The activity of GPDH was stimulated by low pH, a high concentration of glycerol and salt presence in the medium, which is in accordance with earlier literature reports (Adler et al., 1985; Albertyn et al., 1994). In case of D. hansenii, the activity of NADP-dependent GPDH was fivefold higher when the yeast was grown in the presence of 1 M NaCl, compared to media without salt (Alba-Lois et al., 2004). It was also reported that NaCl addition induced the activity of GPDH in the cells of Saccharomyces cerevisiae (Albertyn et al., 1994). The activities of GK and GPDH determined in the cells of Wratislavia K1 strain suggested that glycerol assimilation was the most effective in the media without salt and at higher pH (4.5). The synthesis of erythritol could possibly be due to TK, catalyzing the reaction between fructose-6-phosphate (forming under gluconeogenic conditions) and glyceraldehyde-3-phosphate (interconversion of dihydroxyacetone phosphate). Following this steps, the C4 (erythrose4-phosphate) and C5 (xylulose-5-phosphate) compounds could be dephosphorylated and reduced to erythritol and arabitol as verified in C. magnoliae (Park et al., 2005). It is worth emphasizing that the potential pathway of erythritol formation from glycerol by Y. lipolytica (Fig. 1) has not been investigated to date. However, results presented in this study confirm that TK and ER are involved in the process. In this study, low pH and NaCl addition stimulated the activity of TK. Worthy of notice is that the highest activity of the enzyme, obtained at pH 3.0 and with NaCl presence, was correlated with the highest erythritol concentration obtained (64.0 g L 1) in that culture. In literature, there is a lack of information regarding pH and NaCl influence on TK activity in yeast cells. The TK activity was investigated by Sawada et al. (2009) in the cells of two strains of Trichosporonoides megachiliensis ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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with different capability of producing erythritol. The enzyme activity in the effective phase of erythritol formation was linked with the amounts of erythritol produced. In the cultures with Y. lipolytica Wratislavia K1, the stimulatory effect on ER activity was observed in the case of salt addition. Among many factors examined by Park et al. (2011), NaCl was reported to be the one of the strongest factors influencing ER activity in the cells of C. magnoliae JH110. The presence of salt (23.2 g L 1) resulted in the enzyme activity of 0.050 U mg 1 of protein. However, it needs to be mentioned that the yeast was exposed to the stress factor only for 1 h, and not for the entire process as in the present study. It is worth noting that in all the cultures with Wratislavia K1 strain, the ER activity was lower in the stationary than in the growth phase, which was probably linked to osmotic pressure that was decreasing throughout the process. Therefore, the obtained results confirmed the findings of Park et al. (2011) who observed that ER activity was increasing almost in proportion to the increase of osmotic pressure, which was caused by the addition of sugar. In comparison with other cultures, the activity of CS was the lowest in conditions promoting the citric acid production – at pH 4.5 in media without NaCl addition. Significantly higher activity of CS, up to 0.805 U mg 1 of protein, was obtained in other variants. Hattori & Suzuki (1974b) demonstrated that pH had the significant impact on CS activity and amounts of citrate produced from n-alkanes by C. zeylanoides. During the citrate fermentation conducted at pH 5.5, the CS activity was three times higher (13.2 U mg 1 of protein) when compared to the biosynthesis of erythritol which was performed at pH 3.5. Significantly lower activity of CS (0.820–0.900 U mg 1 of protein) was reported for citrate biosynthesis from glycerol by Y. lipolytica VKM Y-2373 at pH 5.0. Taking into account the high concentration of citric acid obtained in the culture at pH 4.5 without salt and the fact that citrate production is determined by the activity of CS, it might be assumed that the enzyme activity in the cells of Y. lipolytica Wratislavia K1 was inhibited by the increasing concentration of the final product, which is in accordance with literature data (Finogenova et al., 1986). In conclusion, the examined acetate-negative mutants of Y. lipolytica were able to produce high amounts of citric acid from glycerol at pH 5.0–5.5. The cells of Wratislavia AWG7 and Wratislavia 1.31 yeast strains produced more citric acid and excreted it more actively than Wratislavia K1 cells. Lower pH resulted in the inhibition of citric acid and simultaneous stimulation of erythritol formation. The highest amount of erythritol, irrespectively of pH value, was produced by Wratislavia K1. Significant differences in the overproduction of erythritol in the cells of Wratislavia K1 were probably related with the ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

inability of this strain to utilize this polyol. It was demonstrated that the addition of NaCl as well as the use of crude glycerol (which originally contained some amount of the salt) significantly enhanced erythritol production. Moreover, the presence of salt in the medium improved not only erythritol synthesis but also the selectivity of biosynthesis. Furthermore, it was found that in the cells of Y. lipolytica, the enzymes of pentose–phosphate pathways – transketolase and erythrose reductase – were involved in the process of erythritol biosynthesis. The activities of these enzymes were found to be stimulated by low pH and NaCl presence in the medium.

Acknowledgements This work was supported by grant No. N N312 256640 from the National Science Centre (Poland) and by the Ministry of Sciences and Higher Education of Poland and European Union under Project No. POIG 01.01.0200-074/09.

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A comparative study on glycerol metabolism to erythritol and citric acid in Yarrowia lipolytica yeast cells.

Citric acid and erythritol biosynthesis from pure and crude glycerol by three acetate-negative mutants of Yarrowia lipolytica yeast was investigated i...
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