Erythritol Biosynthesis Research Articles

Exploration of Trichoderma reesei as an alternative host for erythritol production

BackgroundErythritol, a natural polyol, is a low-calorie sweetener synthesized by a number of microorganisms, such as Moniliella pollinis. Yet, a widespread use of erythritol is limited by high production costs due to the need for cultivation on glucose-rich substrates. This study explores the potential of using Trichoderma reesei as an alternative host for erythritol production, as this saprotrophic fungus can be cultivated on lignocellulosic biomass residues. The objective of this study was to evaluate whether such an alternative host would lead to a more sustainable and economically viable production of erythritol by identifying suitable carbon sources for erythritol biosynthesis, the main parameters influencing erythritol biosynthesis and evaluating the feasibility of scaling up the defined process.ResultsOur investigation revealed that T. reesei can synthesize erythritol from glucose but not from other carbon sources like xylose and lactose. T. reesei is able to consume erythritol, but it does not in the presence of glucose. Among nitrogen sources, urea and yeast extract were more effective than ammonium and nitrate. A significant impact on erythritol synthesis was observed with variations in pH and temperature. Despite successful shake flask experiments, the transition to bioreactors faced challenges, indicating a need for further scale-up optimization.ConclusionsWhile T. reesei shows potential for erythritol production, reaching a maximum concentration of 1 g/L over an extended period, its productivity could be improved by optimizing the parameters that affect erythritol production. In any case, this research contributes valuable insights into the polyol metabolism of T. reesei, offering potential implications for future research on glycerol or mannitol production. Moreover, it suggests a potential metabolic association between erythritol production and glycolysis over the pentose phosphate pathway.

Enhanced Production of Erythritol from Glucose by the Newly Obtained UV Mutant Yarrowia lipolytica K1UV15.

Erythritol is a polyol with a sweet taste but low energy value. Thanks to its valuable properties, as well as growing social awareness and nutritional trends, its popularity is growing rapidly. The aim of this study was to increase the effectiveness of erythritol production from glucose using new UV mutants of the yeast Yarrowia lipolytica obtained in the Wratislavia K1 strain. The ability of the new strains to biosynthesize erythritol and utilize this polyol was examined in shake-flask cultures and fed-batch processes conducted in a stirred tank reactor with a total glucose concentration of 300 and 400 g/L. The Wratislavia K1 strain produced erythritol most efficiently (97.5 g/L; 192 h) at an initial glucose concentration of 250 g/L (total: 300 g/L). New strains were assessed under such conditions, and it was noted that the highest erythritol concentration (145 g/L; 183 h) was produced by the K1UV15 strain. A significant improvement in the erythritol biosynthesis efficiency (148 g/L; 150 h) was achieved upon the increase in (NH4)2SO4 to 3.6 g/L. Further, in the culture with such a concentration of the nitrogen source and increased total glucose level (400 g/L), the K1UV15 strain produced 226 g/L of erythritol within 281 h.

Application of New Yarrowia lipolytica Transformants in Production of Citrates and Erythritol from Glycerol.

Citric acid and erythritol are obtained on an industrial scale using biotechnological methods. Due to the growing market demand for these products, research is underway to improve the process economics by introducing new microorganisms, in particular of the species Yarrowia lipolytica. The aim of this study was to evaluate transformants of Y. lipolytica for growth and ability to overproduce citric acids and erythritol from glycerol. The transformants were constructed by overexpressing glycerol kinase, methylcitrate synthase and mitochondrial succinate-fumarate transporter in the mutant Wratislavia 1.31. Next, strains were assessed for biosynthesis of citrate (pH 5.5; nitrogen limitation) and erythritol (pH 3.0; high osmotic pressure) from glycerol. Regardless of culture conditions strains, 1.31.GUT1/6 and 1.31.GUT1/6.CIT1/3 exhibited high rates of substrate utilization. Under conditions favoring citrate biosynthesis, both strains produced several percent more citrates, accompanied by higher erythritol production compared to the parental strain. During erythritol biosynthesis, the strain 1.31.GUT1/6.CIT1/3.E34672g obtained as a result of co-expression of all three genes stood out, producing 84.0 g/L of erythritol with yield and productivity of 0.54 g/g and 0.72 g/Lh, respectively, which places it in the group of the highest-ranked producers of erythritol among Y. lipolytica species.

Studies of polyol production by the yeast Yarrowia lipolytica growing on crude glycerol under stressful conditions

Crude glycerol, the principal by-product of biodiesel production process, was employed as substrate by three wild-type Yarrowia lipolytica strains (ACA-YC 5030, LMBF 20 and NRRL Y-323). Stressful conditions (low pH value = 2.0 ± 0.3, low incubation temperature T = 20 ± 1 °C, non-aseptic conditions) were employed. Interesting production of yeast biomass and polyols (viz. erythritol, mannitol and arabitol) was noted at pH = 2.0 ± 0.3 and T = 20 ± 1 °C. Strains failed to produce significant quantities of cellular lipid, while variable quantities of intra-cellular polysaccharides were produced. Fermentations under previously pasteurized media supported significant biomass and polyols production for most of the tested strains, while only one strain (NRRL Y-323), managed to produce polyols at media that were not previously thermally treated at all. The production of mannitol was favored at low initial glycerol (Glol0) concentrations, whereas higher Glol0 quantities favored the biosynthesis of erythritol. For the strain NRRL Y-323, highly aerated / agitated bioreactor trials showed different physiological profiles as compared to the respective flask experiments. Finally, in flask experiments with the strain NRRL Y-323 at high Glol0 amounts (≈140 g/L) at low medium pH (=2.0 ± 0.3), a significant production of polyols (=84.2 g/L) with the corresponding remarkable conversion yield on glycerol consumed = 62 % w/w was achieved. Practical applicationRenewable and biodegradable fuels, such as biodiesel, are safer and environmentally friendlier than the conventional petroleum diesel. Glycerol is a cost-effective substrate obtained as the main side-product from biodiesel production process and is currently being employed in the realm of Industrial Microbiology and Biotechnology to produce metabolic products with added value. Current research focuses on using glycerol as a starting substrate for biotechnological conversions aiming at producing, amongst other compounds, polyols, microbial biomass, citric acid, etc. from selected strains of the Generally Recognized Αs Safe (GRAS) yeast Yarrowia lipolytica. In the current investigation therefore, we examined the capacity of new wild-type non-extensively studied strains of this yeast to grow and assimilate this inexpensive substrate. Specifically, we have performed the acclimatization of the mentioned strains to stressful environments (i.e., low pH, low incubation temperature, non-aseptic conditions, etc.) and remarkable quantities of the added-value compounds (polyols, yeast mass, citric acid) were produced.

Multiplex modification of Yarrowia lipolytica for enhanced erythritol biosynthesis from glycerol through modularized metabolic engineering.

Erythritol is a novelty 4-carbon sugar polyol and has great potential to be used as the precursor of some platform chemicals. The increasing cost of glucose poses researchers shifting insights to the cheaper biodiesel raw materials. Herein, we engineered a non-degradation, non-byproducts Yarrowia lipolytica for the erythritol production with high-titer from glycerol. Initially, the degradation and competition modules were blocked by URA3 counter-selection marker. Subsequently, a shortened biosynthetic pathway was explored to elevate its synthetic flux by multi-modules combination expression of functional genes. Furthermore, a screened glycerol transporter ScFPS1 was integrated into ERY6 genome to promote the glycerol uptake. The constructed strain ERY8 produced 176.66g/L erythritol in the 5-L bioreactor with a yield and productivity of 0.631g/g and 1.23g/L/h, respectively, which achieved the highest fermentation production efficiency till date. This study proposed a novel multi-modules combination strategy for effectively engineering Y. lipolytica to produce erythritol using glycerol.

Understanding the role of GRE3 in the erythritol biosynthesis pathway in Saccharomyces uvarum and its implication in osmoregulation and redox homeostasis.

Erythritol is produced in yeasts via the reduction of erythrose into erythritol by erythrose reductases (ERs). However, the genes codifying for the ERs involved in this reaction have not been described in any Saccharomyces species yet. In our laboratory, we recently showed that, during alcoholic fermentation, erythritol is differentially produced by Saccharomyces cerevisiae and S. uvarum species, the latter being the largest producer. In this study, by using BLAST analysis and phylogenetic approaches the genes GRE3, GCY1, YPR1, ARA1 and YJR096W were identified as putative ERs in Saccharomyces cerevisiae Then, these genes were knocked out in our S. uvarum strain (BMV58) with higher erythritol biosynthesis compared to control S. cerevisiae wine strain, to evaluate their impact on erythritol synthesis and global metabolism. Among the mutants, the single deletion of GRE3 markedly impacts erythritol production, although ΔYPR1ΔGCY1ΔGRE3 was the combination that most decreased erythritol synthesis. Consistent with the increased production of fermentative by-products involved in redox balance in the Saccharomyces uvarum strain BMV58, erythritol synthesis increases at higher sugar concentrations, hinting it might be a response to osmotic stress. However, the expression of GRE3 in the S. uvarum strain was found to peak just before the start of the stationary phase, being consistent with the observation that erythritol increases at the start of the stationary phase, when there is low sugar in the medium and nitrogen sources are depleted. This suggests that GRE3 plays its primary function to help the yeast cells to maintain the redox balance during the last phases of fermentation.

Nitrogen as the major factor influencing gene expression in Yarrowia lipolytica

Yarrowia lipolytica is an important industrial microorganism used for the production of oleochemicals. The design of effective biotechnological processes with this cell factory requires an in-depth knowledge of its metabolism. Here we present a transcriptomic study of Y. lipolytica grown in the presence of glycerol and glucose, and mixture of both at different carbon to nitrogen ratios. It emerged that the transcriptomic landscape of Y. lipolytica is more sensitive to the nitrogen availability than to the utilized carbon source, as evidenced by more genes being differentially expressed in lower carbon to nitrogen ratio. Specifically, expression of hexokinase (HXK1) is significantly susceptible to changes in nitrogen concentrations. High HXK1 expression in low nitrogen seems to impact other genes which are implicated in tricarboxylic acid cycle and erythritol biosynthesis. We further show that expression of HXK1 and two genes belonging to the sugar porter family might be controlled by GATA-like zinc-finger proteins.

Erythritol: Another C4 Platform Chemical in Biomass Refinery.

The potential of erythritol as a platform chemical in biomass refinery is discussed in terms of erythritol production and utilization. Regarding erythritol production, fermentation of sugar or starch has been already commercialized. The shift of the carbon source from glucose to inexpensive inedible waste glycerol is being investigated, which will decrease the price of erythritol. The carbon-based yield of erythritol from glycerol is comparable to or even higher than that from glucose. The metabolic pathway of erythritol biosynthesis has become clarified: erythrose-4-phosphate, which is one of the intermediates in the pentose phosphate pathway, is dephosphorylated and reduced to erythritol. The information about the metabolic pathway may give insights to improve the productivity by bleeding. Regarding erythritol utilization, chemical conversions of erythritol, especially deoxygenation, have been investigated in these days. Erythritol is easily dehydrated to 1,4-anhydroerythritol, which can be also used as the substrate for production of useful C4 chemicals. C–O hydrogenolysis and deoxydehydration using heterogeneous catalysts are effective reactions for erythritol/1,4-anhydroerythritol conversion.

Unexpected roles for ADH1 and SORD in catalyzing the final step of erythritol biosynthesis

The low-calorie sweetener erythritol is endogenously produced from glucose through the pentose phosphate pathway in humans. Erythritol is of medical interest because elevated plasma levels of this polyol are predictive for visceral adiposity gain and development of type 2 diabetes. However, the mechanisms behind these associations remain unknown because the erythritol biosynthesis pathway, particularly the enzyme catalyzing the final step of erythritol synthesis (reduction of erythrose to erythritol), is not characterized. In this study, we purified two enzymes from rabbit liver capable of catalyzing the conversion of erythrose to erythritol: alcohol dehydrogenase 1 (ADH1) and sorbitol dehydrogenase (SORD). Both recombinant human ADH1 and SORD reduce erythrose to erythritol, using NADPH as a co-factor, and cell culture studies indicate that this activity is primarily NADPH-dependent. We found that ADH1 variants vary markedly in both their affinity for erythrose and their catalytic capacity (turnover number). Interestingly, the recombinant protein produced from the ADH1B2 variant, common in Asian populations, is not active when NADPH is used as a co-factor in vitro We also confirmed SORD contributes to intracellular erythritol production in human A549 lung cancer cells, where ADH1 is minimally expressed. In summary, human ADH1 and SORD catalyze the conversion of erythrose to erythritol, pointing to novel roles for two dehydrogenase proteins in human glucose metabolism that may contribute to individual responses to diet. Proteomics data are available via ProteomeXchange with identifier PXD015178.

Heterologous overexpression of bacterial hemoglobin VHb improves erythritol biosynthesis by yeast Yarrowia lipolytica

BackgroundYarrowia lipolytica is an unconventional yeast with a huge industrial potential. Despite many advantages for biotechnological applications, it possesses enormous demand for oxygen, which is a bottleneck in large scale production. In this study a codon optimized bacterial hemoglobin from Vitreoscilla stercoraria (VHb) was overexpressed in Y. lipolytica for efficient growth and erythritol synthesis from glycerol in low-oxygen conditions. Erythritol is a natural sweetener produced by Y. lipolytica under high osmotic pressure and at low pH, and this process requires high oxygen demand.ResultsUnder these conditions the VHb overexpressing strain showed mostly yeast-type cells resulting in 83% higher erythritol titer in shake-flask experiments. During a bioreactor study the engineered strain showed higher erythritol productivity (QERY = 0.38 g/l h) and yield (YERY = 0.37 g/g) in comparison to the control strain (QERY = 0.30 g/l h, YERY = 0.29 g/g). Moreover, low stirring during the fermentation process resulted in modest foam formation.ConclusionsThis study showed that overexpression of VHb in Y. lipolytica allows for dynamic growth and efficient production of a value-added product from a low-value substrate.

Kinetic Study of Erythrose Reductase Extracted from Yarrowia lipolytica

Erythritol as a non-caloric and non-cariogenic sweetener is safe for diabetics. Both microbial fermentation and chemical methods can be used to produce erythritol, but chemical methods failed to be industrialized due to their low efficiency. Moniliella tomentosa, Aureobasidium sp. and Yarrowia lipolytica are industrial producers of erythritol. Erythrose reductase (ER) is a key enzyme in the biosynthesis of erythritol and catalyzes the final step in this pathway. Enzyme extract was obtained from Y. lipolytica by grinding cells with 0.5mm glass beads and ER activity was performed using 10 µl enzyme extract, 7.5 mM NADPH and 12 mM D-erythrose in potassium phosphate buffer (pH 7.5). Reaction was monitored with decreasing of NADPH absorbance in OD340 at 37 ˚C for 8 min by a microplate analyzer. In order to determine the activation energy (Ea), activity of enzyme was measured in 4-45 ˚C and results were analyzed with Kinetic software according to Arrhenius equation. The best enzyme activity of ER was 6.268 mU. One unit of ER activity was defined as the amount of enzyme that catalyzes the oxidation of 1μmol of NADPH per minute. Specific activity of enzyme was equal to 3.24U/mg and finally the Ea was determined to be 29.6208 KJ.ER specific activity in this study was lower than the only similar study that used Y. lipolytica. Purification, overexpression and optimizing the reaction can help to increase enzyme performance.

Effects of osmotic pressure and pH on citric acid and erythritol production from waste cooking oil by Yarrowia lipolytica.

Erythritol and citric acid could be produced from waste cooking oil (WCO) by Yarrowia lipolytica under different medium conditions, and osmotic pressure together with pH were considered to be the critical factors in this process. High osmotic pressure (2.76osmol/L) combined with low pH (pH 3.0) promoted the highest yield of erythritol (21.8g/L) accompanied by low-producing citric acid (2.5g/L). By contrast, the highest citric acid biosynthesis (12.6g/L) was detected under a pH of 6.0 and an osmotic pressure of 0.75osmol/L, when only 4.0g/L of erythritol was yielded. Moreover, lipase activities in these two media were also detected, and pH 3.0-OP 2.76 was supposed to be more beneficial to lipase activity. Biochemical pathways involved in the biosynthesis of erythritol and citric acid were subsequently investigated, and the products yielded from WCO were assumed to be correlated with the activities of transketolase, erythrose reductase, citrate synthase, and glycerol kinase. However, RT-PCR analysis revealed that mRNA levels of these enzymes did not significantly differ, confirming that metabolic flux regulations of erythritol and citric acid mostly took place at the post-transcriptional level.

Enhancement of erythritol production by Trichosporonoides oedocephalis ATCC 16958 through regulating key enzyme activity and the NADPH/NADP ratio with metal ion supplementation

ABSTRACTErythritol, a well-known natural sweetener, is mainly produced by microbial fermentation. Various metal ions (Al3+, Cu2+, Mn2+, and Ni2+) were added to the culture medium of Trichosporonoides oedocephalis ATCC 16958 at 30 mg/L in shake flask cultures. Compared with controls, Cu2+ increased the erythritol content by 86% and decreased the glycerol by-product by 31%. After 48 hr of shake flask culture, sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that expression levels of erythrose reductase (ER) in the presence of 30 mg/L CuSO4 · 5H2O were higher than those obtained after treatment with other examined metal ions. Furthermore, after 108 hr of batch culture in a 5-L bioreactor, supplementation with 30 mg/L of CuSO4 · 5H2O increased the specific erythritol content by 27%. Further studies demonstrated that ER activity under 30 mg/L CuSO4 · 5H2O supplementation in a fermentor was overtly increased compared with the control after 60 hr, while glycerol-3-phosphate dehydrogenase activity was clearly reduced in most of the fermentation process. Furthermore, the NADPH/NADP ratio was slightly lower in T. oedocephalis cells treated with Cu2+ compared with control cells. These results provide further insights into Cu2+ effects on erythritol biosynthesis in T. oedocephalis and should improve the industrial production of erythritol by biological processes.

Two-stage continuous culture – Technology boosting erythritol production

Yield and product titer are two important parameters for economic evaluation of any biotechnological process. The published studies on erythritol biosynthesis are based mainly on batch and fed-batch cultures, which do not fully utilize the production potential of the cells. In the current work, a two-stage continuous process of erythritol production from glycerol by an industrially relevant strain of Yarrowia lipolytica is proposed. The proposed process significantly enhanced titer of secreted erythritol and reduced process costs compared to the conventional batch cultures on glycerol. The two-stage chemostat process with glycerol resulted in 199.4 g dm−3 of erythritol with overall yield of 0.66 g g−1 and productivity of 0.8 g dm−3 h−1. These results represent almost 2.5-fold higher titer of erythritol compared to commonly used batch cultures of Y. lipolytica and almost 2 times higher titer and 1.3-fold increase in the product yield compared to a previously published continuous process with glycerol as a substrate. This study showed the potential of a two-stage continuous process using a genetically unmodified strain of Y. lipolytica for efficient erythritol production from raw glycerol originating from different industries.

A cost-effective process for the coproduction of erythritol and lipase with Yarrowia lipolytica M53 from waste cooking oil

Waste cooking oil (WCO), an oil-based substance that is produced from cooking, causes heavy burden to the environment and society. The coproduction of erythritol and lipase by Yarrowia lipolytica from this low-cost fatty matter was evaluated in this study. Compared with other oily substances, WCO was more favorable for erythritol and lipase coproduction, and the optimum concentration was 30.0g/L. As an osmotic agent, 80.0g/L of NaCl was required for the efficient biosynthesis of erythritol. The beneficial nitrogen source for the coproduction was ammonium oxalate, with a C/N ratio of 87:1. Moreover, key enzymes for erythritol synthesis were induced in WCO medium. The coproduction was then scaled up in a 5L fermenter. The maximum concentration of erythritol (22.1g/L) was obtained after 72h, corresponding to a yield of 0.74g/g, whereas the highest lipase activity reached 12.7U/mL in 24h. This study proposed a low-cost bioprocess for synthesizing high-value products using WCO.

Complementary function of two transketolase isoforms from Moniliella megachiliensis in relation to stress response

Two transketolase isogenes, MmTKL1 and MmTKL2, isolated from Moniliella megachiliensis were investigated for their roles in stress response and erythritol biosynthesis. The encoded proteins were highly homologous in amino acid sequence and domain structure. Two stress response elements (STREs) were found upstream of MmTKL1, while no STRE was found upstream of MmTKL2. In contrast, two Ap-1 elements were present upstream of MmTKL2, but none were detected upstream of MmTKL1. MmTKL2 partially complemented the aromatic amino acid auxotrophy of a Saccharomyces cerevisiae tkl1 deletion mutant, suggesting that at least one of the MmTKLs functioned as a transketolase in vivo. In response to short-term osmotic stress (20% glucose or 1.2 M NaCl) in Moniliella cells, MmTKL1 expression increased rapidly through the first 40 min before subsequently decreasing gradually, while MmTKL2 expression showed no significant change. In contrast, short-term oxidative stress (0.15 mM menadione) induced considerable increases in MmTKL2, while MmTKL1 expression remained low under the same conditions. Long-term osmotic stress (20% glucose) yielded increased expression of both genes starting at 12 h and continuing through 72 h. During either osmotic or oxidative stress, intracellular erythritol accumulation could clearly be correlated with the pattern of expression of either MmTKL1 or MmTKL2. These results strongly suggested that MmTKL1 is responsible primarily for the response to osmotic stress, while MmTKL2 is responsible primarily for the response to oxidative stress. Thus, we postulate that the two transketolase isoforms of M. megachiliensis play distinct and complementary roles in coordinating erythritol production in response to distinct environmental stresses.

Polyol production during heterofermentative growth of the plant isolate Lactobacillus florum 2F.

This study examined the fermentative growth and polyol production of Lactobacillus florum and other plant-associated lactic acid bacteria (LAB). Sugar consumption and end-product production were measured for Lact.florum 2F in the presence of fructose, glucose and both sugars combined. The genome of Lact.florum was examined for genes required for mannitol and erythritol biosynthesis. The capacity for other plant-associated LAB to synthesize polyols was also assessed. Lactobacillus florum exhibited higher growth rates and cell yields in the presence of both fructose and glucose. Lactobacillus florum 2F produced lactate, acetate and ethanol as well as erythritol and mannitol. Lactobacillus florum 2F synthesized mannitol during growth on fructose and erythritol during growth on glucose. Gene and protein homology searches identified a mannitol dehydrogenase in the Lact.florum 2F genome but not the genes responsible for erythritol biosynthesis. Lastly, we found that numerous other heterofermentative LAB species synthesize erythritol and/or mannitol. Lactobacillus florum is a recently identified, plant-associated, fructophilic LAB species. Our results show that Lact.florum growth rates and heterofermentation end-products differ depending on the sugar substrates present and growth yields can be improved when combinations of sugars are provided. Lactobacillus florum 2F produces erythritol and mannitol, two polyols that are relevant to foods and potentially also in plant environments. The capacity for polyol biosynthesis appears to be common among plant-associated, LAB species.

Erythritol biosynthesis from glycerol by Yarrowia lipolytica yeast: effect of osmotic pressure

AbstractThe aim of this study was to examine the impact of osmotic pressure, regulated by an addition of different NaCl concentrations, on the production parameters and activity of the enzymes involved in the biosynthesis of erythritol from glycerol by Yarrowia lipolytica yeast. In the bioreactor batchcultures, strain A-3 was able to produce from 25.3 g dm

Newly isolated mutant of Yarrowia lipolytica MK1 as a proper host for efficient erythritol biosynthesis from glycerol

Mutants of Yarrowia lipolytica Wratislavia K1 were generated by ultraviolet mutagenesis to enhance erythritol production and to minimize by-products formation. One of the mutants named as MK1 was selected for further investigation. In batch culture strain Y. lipolytica MK1 was able to produce up to 82.2gl−1 erythritol with 0.55gg−1 yield and productivity of 0.84gl−1h−1. The concentration of by-products was below 5% of all synthesized metabolites. In order to enhance the production of erythritol and to inhibit the production of by-products the repeated batch culture (RBC) was performed. In RBC strain Y. lipolytica MK1 gave maximum erythritol production 224gl−1 with 0.77gg−1 yield and productivity of 0.54gl−1h−1. The concentration of by-products was below 2.3% of all synthesized metabolites. Moreover, the lipid and fatty acid content in the biomass was determined. This is the first study on newly isolated Y. lipolytica mutant. It demonstrates that Y. lipolytica MK1 is able to produce significantly high erythritol with strong inhibition of by-products formation.

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 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.