ERYTHROSE REDUCTASE AS AN ENZYME RESPONSIBLE FOR ERYTHRITOL OVERPRODUCTION IN YARROWIA LIPOLYTICA YEAST GROWN ON GLYCEROL MEDIA

Ludwika Tomaszewska-Hetman, Anita Rywińska
Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

On the basis of initial screening of Yarrowia lipolytica strains, performed in the shake-flasks experiment using a glycerol medium, three strains with different ability of erythritol production were selected. The yield and dynamics of the biosynthesis by chosen strains were examined in bioreactor cultures and the activities of enzymes responsible for glycerol consumption and erythritol synthesis were analyzed during the process. The comparison of  relationship between erythritol production ability and activities of the examined enzymes indicated erythrose reductase (ER) as the key enzyme responsible for erythritol overproduction in Y. lipolytica cells grown on glycerol. In the culture, the activity of ER in the effective phase of erythritol biosynthesis reached 0.068–0.248 U mg^-1, whereas erythritol production was from 44.7 to 75.9 g L^-1 with the yield of 0.31–0.51 g g^-1. The cells ability of erythritol formation depended slightly on the glycerol kinase and glycerol-3-phosphate dehydrogenase.

Key words: erythritol, glycerol, Yarrowia lipolytica, enzyme activity.

INTRODUCTION

Erythritol is a sugar alcohol and a natural compound of a human diet, occurring in fruits, mushrooms, seaweeds, fermented food and beverages. Although it has only 60–80% of the sweetness of sucrose, it is eagerly employed in food production due to very low caloric value (0–0.2 kcal g^-1) and rheological properties similar to sugar. The application of erythritol is more beneficial than the use of other polyols (e.g. mannitol, sorbitol, xylitol) and artificial sweeteners because it has no bad aftertastes, its consumption is safe and it does not cause gastric side-effects [1, 14].

Erythritol is the only one polyol produced exclusively in microbiological processes because of very low yield and the high costs of the chemical methods of its synthesis [12]. Because of a growing interest in erythritol, especially from the food market, a number of studies have been undertaken to develop the highly-effective erythritol biosynthesis by screening and breeding microorganisms and by optimizing process conditions.  Among the known erythritol-producing microorganisms the most extensively studied were Candida magnoliae, Moniliella sp., Torula corallina and Trichosporonoides megachiliensis [3, 8–10, 13, 18]. The detailed metabolic studies of erythritol production by these species included identification of enzymes, investigation of protein expression and cloning of genes involved in the process.

The most typical substrates for the erythritol biosynthesis, used in laboratory and industrial-scale processes, include glucose, sucrose, fructose or starch hydrolysates [12]. The use of these traditional substrates is inextricably linked to the “food versus fuel” debate, which has socioeconomic implications [4]. This problem does not apply to crude materials, such as glycerol a by-product of biodiesel industry, which has recently been proposed as an alternative substrate for the process of erythritol production by Yarrowia lipolytica yeast [15, 16, 20, 21, 26]. Although the impact of different factors on the erythritol production ability of Y. lipolytica has been reported, the regulatory mechanisms leading to over-production of erythritol from glycerol by Y. lipolytica have not been investigated and explained so far.

The aim of this study was to compare the activity of enzymes involved in glycerol consumption and erythritol formation in the cells of Y. lipolytica strains exhibiting different ability of erythritol biosynthesis, as well as to indicate the enzymes responsible for the polyol overproduction.

MATERIAL AND METHODS

Microorganisms
The strains A-3, A-8, Wratislavia AWG7, Wratislavia 1.31, Wratislavia K1 of Yarrowia lipolytica originated from the yeast culture collection belonging to the Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences in Poland. The strain Y. lipolytica CCY-29-26-5 was from the Czechoslovak Collection of Yeasts. All the strains were stored on YM slants at 4°C.

Media
Growth medium, for seed cultures, consisted of 50.0 g L^-1 of pure glycerol (98%), 3.0 g L^-1 of yeast extract, 3.0 g L^-1 of malt extract and 5.0 g L^-1 of bacto-peptone dissolved in distilled water. Production medium for the shake-flasks experiment contained 100.0 g L^-1 of pure glycerol, 2.0 g L^-1 of NH₄Cl, 1.0 g L^-1 of yeast extract, 0.2 g L^-1 of KH₂PO₄, 1.0 g L^-1 of MgSO₄ x 7H₂O, 3.0 g L^-1 of CaCO₃ and 25.0 g L^-1 of NaCl and distilled water. Optimized production medium for bioreactor cultivations was prepared from 150.0 g L^-1 of pure glycerol, 2.25 g L^-1 of (NH₄)2SO₄, 1.0 g L^-1 of yeast extract, 0.22 g L^-1 of KH₂PO₄, 1.0 g L^-1 of MgSO₄ x 7H₂O, 26.4 g L^-1 of NaCl and tap water. All the media were sterilized for 20 min at 121°C.

Culture conditions
The seed cultures were carried out in 0.3-L flasks containing 0.1 L of growth medium on a rotary-shaker (CERTOMAT IS, Sartorius Stedim Biotech GmbH) at 29.5°C and 140 rpm for 3 days. The shake-flasks experiment was conducted for 10 days in 0.3-L flasks, containing 0.03 L of the appropriate production medium inoculated with 1 mL of seed culture, in the same conditions as described above. The samples for analysis were taken at the end of the experiment. Bioreactor cultures 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, the aeration rate fixed at 0.6 v/v/min and the stirrer speed adjusted to 800 rpm. Production media for bioreactor cultures were inoculated with 0.2 L of the inoculation culture. The pH of 3.0 was maintained automatically by the addition of a 20% (w/v) NaOH solution. Samples were taken 2–3 times per day. Results of experiments are presented as the mean values of three replicates.

Analytical methods
Analysis of the samples was performed according to the earlier-described method [21]. The dry cell weight was determined by the weighing method, whereas the concentrations of substrate (glycerol), product (erythritol), and by-products (arabitol, mannitol, citric and alpha-ketoglutaric acids) were determined by the HPLC method.

Enzyme assays
The activities of enzymes were measured in the growth and at the end of the stationary phases of the cultures. In order to determine the enzyme activities, the sample of 0.2 L was taken from the bioreactor culture and prepared according to the methodology described before [23]. Activities of enzymes: glycerol kinase (EC 2.7.1.30), glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), transketolase (EC 2.2.1.1) and erythrose reductase (EC 1.1.1.21), were determined according to the methods described earlier by Kamzolova et al. [7], White and Kaplan [24], Sugimoto and Shiio [19] and Lee et al. [9], respectively. Protein content of the samples was assayed by Lowry’s method. One unit of the enzyme activity (U) represented 1 micro-mol of NADH/NADPH consumed or produced per 1 minute. The enzyme activities were expressed as specific activity (U per mg of protein) as well as biomass activity (U per g of yeast biomass dry weight).

Statistical analysis
One-way analysis of variance was performed using Statistica 10.0 software (StatSoft, Tulsa, OK, USA) to detect significant differences in the data (biomass and erythritol production) depending on the yeast strain. Homogeneous groups were determined using Duncan’s test (P ≤ 0.05). The linear regression of enzymes activities and the amount of erythritol produced by the yeast cells was determined (P≤ 0.05) using the software mentioned above.

RESULTS AND DISCUSSION

In the first step of the study, six strains of Yarrowia lipolytica yeast were used for the 10-day shake-flasks experiment in order to compare the capability of yeast to produce erythritol when grown in glycerol medium. The culture medium was enriched with NaCl and set up at pH 3.0 as our previous studies showed that the sodium chloride presence and low pH of media promoted erythritol production by Y. lipolytica [15, 21]. After the cultivation it was observed that the biomass level depended on the yeast strain and ranged from 7.7 to 11.2 g L^-1 (Fig. 1). Although all the examined strains were able to produce erythritol, the final concentrations of the polyol were significantly different and varied from 9.5 to 32.9 g L^-1. The best producing strain was found to be Wratislavia K1, application of which allowed to obtain 32.9 g L^-1 of erythritol with the yield of 0.33 g g^-1. In contrast, in the cultures with strain A-3 the final erythritol concentration and production yield reached only 9.5 g L^-1 and 0.11 g g^-1, respectively.

Different effectiveness of product biosynthesis might be linked to the various level of activities of enzymes. In order to evaluate  relationship between erythritol production and activities of enzymes involved in the biosynthesis pathway for further experiments three strains with different potential of erythritol production were selected. On the basis of shake-flasks experiment the following strains were chosen: Wratislavia K1 - indicated as the best producing strain, CCY 29-26-5 and A-3. In comparison to Wratislavia K1, the obtained amount of erythritol and its production yield in the cultures of CCY 29-26-5 and A-3 strains reached about 70 and 30%, respectively.

In order to confirm different abilities of erythritol production by selected strains, in the next step bioreactor production cultures with optimized medium were performed, as it is known that an improvement of biosynthesis parameters might be obtained in bioreactors [17]. During cultivations, the  concentration of substrates and products was monitored and the cultures were cultivated until the time of complete exhaustion of glycerol from the medium. The final results of the cultivations are presented in Table 1. Depending on the strain, the biosynthesis lasted 83.5 to 95 h and biomass production after the process reached 14.8–20.0 g L^-1. A large variation of erythritol synthesis ability between strain A-3, CCY 29-26-5 and Wratislavia K1 was observed, as the final polyol concentrations in the culture broths with these strains reached 44.7, 52.6 and 75.9 g L^-1, respectively, and were comparable with earlier reports [16, 21]. The values of erythritol production parameters, such as yield and volumetric productivity, depended on the applied strain and were at the level of 0.31–0.51 g g^-1 and 0.49–0.80 g L^-1h^-1, respectively. The results indicated the same relation as was observed before.  The highest parameters of the biosynthesis were achieved with the use of Wratislavia K1, whereas CCY 29-26-5 showed moderate ability of erythritol production, and A-3 was found to be the weakest producing strain. In bioreactor cultures the amount of produced erythritol in the cultures of CCY 29-26-5 and A-3 strains reached about 70 and 60% of the amount produced by Wratislavia K1, respectively. Compared to the results obtained from the shake-flasks experiment, the increase of erythritol production was noted for strains A-3 and CCY 29-26-5, which was probably caused by better control of biosynthesis parameters (such as aeration and pH) in the bioreactor. Apart from erythritol, by-products synthesis was observed in the bioreactor cultures, which was especially high for the culture with strain CCY 29-26-5 where the concentration of mannitol, arabitol and citric acid reached 6.2, 4.3 and 12 g L^-1, respectively. The amount of alpha-ketoglutaric acid was the highest in the culture of A-3 strain where it reached 3.3 g L^-1.

Next, the chosen strains were compared in terms of the activity of enzymes suspected to be involved in the metabolism of glycerol to erythritol (Fig. 2). In order to investigate the activity of chosen enzymes, the samples were withdrawn from the bioreactor cultures during the yeast growth phase and at the end of the stationary phase, just before the glycerol depletion from the culture medium. Glycerol is a small and uncharged molecule therefore it might cross the cytoplasmic membrane through passive diffusion. However, it was also reported that it may enter microbial cells by facilitated diffusion, achieved by an integral membrane protein, or by an active uptake [2]. Next, intracellular glycerol might be metabolized via two possible pathways, by the phosphorylation or oxidation. However, the investigation on Y. lipolytica cells showed that the yeast used only the phosphorylation pathway, in which glycerol kinase (GK) catalyzes glycerol phosphorylation to glycerol-3-phosphate which is subsequently converted via dehydrogenation by glycerol-3-phosphate dehydrogenase (GPDH) to dihydroxyacetone-phosphate [2, 5, 11, 25] that might be integrated into different pathways resulting in biomass, lipids, citric, pyruvic or alpha-ketoglutaric acids formation [16]. In the presented work, the activities of GK and GPDH, enzymes responsible for glycerol utilization, were determined in the prepared cell extract and presented in Table 2.  In the case of the best erythritol-producing strain, Wratislavia K1, the activity of GK was found to be higher than in the cells of the weakest producer, A-3 strain, irrespectively of the culture phase.  The correlation was observed between the enzyme activity in the yeast growth phase, calculated per yeast DW, and the yeast growth observed in the culture broths (Tab. 1). Moreover, it was noted that the activity of GK decreased during the cultivation time, which was probably caused by decreasing concentration of glycerol in the culture medium, as GK activity might be induced by the substrate [11]. For all examined strains, the specific activity of GPDH determined in the yeast growth phase was at a similar level of 0.011–0.015 U mg^-1. In the cells of A-3 strain, the enzyme activity did not change, whereas for other strains it increased twofold in the stationary phase (Tab. 2).

Up to date, the mechanisms of the erythritol biosynthesis has been studied in terms of its formation from glucose. Two different pathways were proposed for bacteria and yeast. In bacteria, glucose-6-phosphate is converted by phosphoglucose isomerase to fructose-6-phosphate which is then cleaved to erythrose-4-phosphate by phosphoketolase. Next, the reduction of erythrose-4-phosphate is catalyzed by erythritol-4-phosphate dehydrogenase and the hydrolysis of erythritol-4-phosphate to erythritol by phosphatase takes place. In turn, in yeast, glucose-6-phosphate is metabolized via pentose-phosphate pathway (Fig. 2). The intermediate of this pathway, erythrose-4-phosphate is a precursor for nucleotide and amino acids biosynthesis but it might also undergo dephosphorylation to erythrose which is then reduced by erythrose reductase (ER) to erythritol [12]. As for the yeast, two enzymes were suggested to have the greatest impact on erythritol production from glucose. Transketolase (TK), an enzyme from pentose-phosphate pathway, was reported to play the crucial role in erythritol production by T. megachiliensis [18], whereas ER was indicated as the key enzyme when Aureobasidium sp. and C. magnoliae were applied in the process [6, 10]. In this study, the highest specific activity of TK was determined in the cells of Wratislavia K1 strain, in the yeast growth phase, where it reached 0.081 U mg^-1 (Tab. 2). Generally, it was observed that the activity of TK decreased during the course of the culture. An exception was the A-3 strain which performed comparatively high specific activity of TK during the cultivation time. It should be noted that high activity of the TK does not have to be unambiguously related to effective erythritol formation because, as mentioned before, the intermediates from pentose-phosphate pathway are used for the biosynthesis of a number of other compounds. Among the examined strains significant differences were observed in the ER activity, especially in stationary phase of the culture (Tab. 2). In the case of the weakest erythritol producer (A-3) both, the specific activity and the activity of the biomass of ER dropped significantly during the process, whereas for the best erythritol-producing strain (Wratislavia K1) a significant increase was noted in the enzyme activity. In the stationary phase, therefore in the phase of the effective erythritol production, ER activity reached 0.068, 0.212, 0.248 U mg^-1, respectively, for strains A-3, CCY 29-26-5 and Wratislavia K1 and was correlated with the amounts of erythritol produced by the strains (Tab. 1).

In order to elucidate the effect of activities of the chosen enzymes on erythritol overproduction ability, the obtained results were compared with the amounts of the polyol produced by the yeast cells (Fig. 3). In the yeast growth phase, enzymes activities did not contribute to enhanced erythritol production (Fig. 3A). However, in the effective production phase (Fig. 3B) the cells ability of erythritol formation depended slightly on the GK and GPDH but principally on the ER activity. Moreover, it might be noticed that high overproduction ability shown by Wratislavia K1 strain (Tab. 1) seemed to be correlated with the activity of ER (Tab. 2, Fig. 4) which did not decrease but remained at a high level throughout the cultivation time.

CONCLUSION

In the last decade, the price of glycerol has been continuously decreasing, which increased the interest in using glycerol as a low-cost substrate for biotechnological processes. The biosynthesis of erythritol from glycerol by Y. lipolytica is an alternative approach to the polyol production – environmentally-friendly and cost-effective. Recently, the literature reported about different factors affecting the formation of erythritol from glycerol by Y. lipolytica which will lead to the development and optimization of the industrially-competitive process. In the presented study, the metabolic aspects of glycerol conversion to erythritol in the yeast cells were studied in order to clarify and understand the process. Y. lipolytica strains were selected and compared in terms of erythritol production ability and activities of the enzymes involved in glycerol utilization and erythritol formation. The results obtained clearly indicated that in the biosynthesis of erythritol from glycerol in the cells of Y. lipolytica the key enzyme, responsible for erythritol overproduction was erythrose reductase.

ACKNOWLEDGMENTS

This work was sponsored by grant No. N N312 256640 from the National Science Centre (Poland).

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Accepted for print: 16.11.2015

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