Erythritol Production Research Articles

Functional overexpression of genes involved in erythritol synthesis in the yeast Yarrowia lipolytica

BackgroundErythritol, a four-carbon polyol synthesized by microorganisms as an osmoprotectant, is a natural sweetener produced on an industrial scale for decades. Despite the fact that the yeast Yarrowia lipolytica has been reported since the 1970s as an erythritol producer, the metabolic pathway of this polyol has never been characterized. It was shown that erythritol synthesis in yeast occurs via the pentose phosphate pathway (PPP). The oleaginous yeast Y. lipolytica is a good host for converting inexpensive glycerol into a value-added product such as erythritol. Glycerol is a renewable feedstock which is produced on a large scale as a waste product by many branches of industry.ResultsIn this study, we functionally overexpressed four genes involved in the pentose phosphate pathway (PPP): gene YALI0E06479g encoding transketolase (TKL1), gene YALI0F15587g encoding transaldolase (TAL1), gene YALI0E22649g encoding glucose-6-phosphate dehydrogenase (ZWF1), and gene YALI0B15598g encoding 6-phosphogluconate dehydrogenase (GND1). Here, we show that the crucial gene for erythritol synthesis in Y. lipolytica is transketolase. Overexpression of this gene results in a twofold improvement in erythritol synthesis during a shake-flask experiment (58 g/L). Moreover, overexpression of TKL1 allows for efficient production of erythritol independently from the supplied dissolved oxygen. Fermentation conducted in a 5-L bioreactor at low agitation results in almost 70% higher titer of erythritol over the control strain.ConclusionThis work presents the importance of the PPP in erythritol synthesis and the feasibility for economic production of erythritol from glycerol by the yeast Y. lipolytica.

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.

An Effective Method of Continuous Production of Erythritol from Glycerol by Yarrowia lipolytica MK1.

This study demonstrates the potential applicability of the UV mutant Yarrowia lipolytica MK1 for the valorisation of glycerol and erythritol production in a chemostat culture. The aim of this research is to investigate the optimal C:N ratio in the feeding medium in order to enhance erythritol production. The highest erythritol concentration, at 113.1 g/L with a volumetric erythritol production rate of 1.1 g/(L·h) and a yield of 0.57 g/g, was obtained in the feeding medium with a C:N ratio of 80:1. Moreover, no residual glycerol was observed in the culture broth during cultivation. The chemical composition of the biomass was analysed. The contents of lysine and threonine in the biomass protein amino acid profile were higher than those required by the FAO/WHO for fodder yeast.

Enhanced production of insect raw-starch-digesting alpha-amylase accompanied by high erythritol synthesis in recombinant Yarrowia lipolytica fed-batch cultures at high-cell-densities

Abstract Recently we reported on raw-starch-digesting ability of alpha-amylase from an insect Sitophilus oryzae (SoAMY) expressed in recombinant Yarrowia lipolytica cells, and demonstrated its usefulness in simultaneous saccharification and fermentation processes with industrial yeasts. In this study we applied fed-batch cultures of Y. lipolytica 4.29 strain reaching high-cell-densities (up to 70 [gDCW/L]), to enhance SoAMY production. SoAMY activity in the medium reached the peak value of 22,979.23 ± 184 [AU/L], at volumetric productivity of 121.58 ± 1.75 [AU/L/h], and yield of 71.83 ± 3.08 [AU/gglycerol], constituting roughly 160-fold improvement, compared to the best previous result. The cultivations were accompanied by high production of erythritol (83.58 [g/L]), at the marginal production of mannitol (5.46 [g/L]). Elementary analyses of media constituents, the enzyme and the yeast biomass gave better insight into carbon and nitrogen fluxes distribution. Due to application of genetic engineering and bioprocess engineering strategies, the insect-derived enzyme can be produced at the quantities competitive to microbial catalysts.

A novel strain of Yarrowia lipolytica as a platform for value-added product synthesis from glycerol.

BackgroundIncreasing interest of non-conventional yeasts has been observed for many years due to their biochemical characteristics and potential applications. Well-studied, oleaginous yeast Y. lipolytica is an attractive host for converting a low-cost glycerol, into value-added products such as erythritol (sweetener) or citric acid. Glycerol is an important renewable feedstock and is the main co-product of biodiesel production, which is nowadays applied on a large commercial scale. To this end, we engineered the yeast Y. lipolytica to increase the productivity of this strain.ResultsIn this light, we enhanced glycerol assimilation by over-expression of the YALI0F00484g gene encoding glycerol kinase (GK) and gene YALI0B02948g encoding glycerol-3-P dehydrogenase (GDH). The modified strains have been tested for glycerol consumption rate and erythritol and citric acid synthesis under various conditions. Here, we show that the overexpression of GK and GDH, increased glycerol consumption resulting in rapid erythritol and citric acid synthesis. Next, we combined the two genes in the tandem gene construct for the simultaneous co-expression of GK and GDH, which further increased the desired product synthesis. The glycerol consumption was explored in a 5-L bioreactor and the engineered strains were able to utilize 150 g/L glycerol within 44–48 hours. The erythritol productivity for GK overexpression and co-expression of GK and DGH was 24 and 35 %, respectively, over the control strain. Moreover, we established conditions for the production of citric acid at pH 3.0, the engineered strains increased citric acid production 14-fold over the control.ConclusionThis work demonstrates the excellent capacity of the engineered strains as a starting platform for further modification for broad-range value-added product biosynthesis from glycerol. This study presents the highest reported titer citric acid at low pH to date. The process parameters such as productivity and yield of erythritol and citric acid were significantly elevated, what is valuable for industrial applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0593-z) contains supplementary material, which is available to authorized users.

Technology of efficient continuous erythritol production from glycerol

Erythritol production in chemostat culture from pure and crude glycerol with different nitrogen sources by Yarrowia lipolytica Wratislavia K1 was investigated. Moreover, a process of erythritol purification based on ion exchange is proposed. The highest erythritol production (103.4 g L−1 with a volumetric erythritol production rate of 1.12 g L−1 h−1 and a yield of 0.52 g g−1) was obtained by applying an inorganic nitrogen source (4.6 g L−1 of ammonium sulfate). Very promising results were also obtained when pure glycerol was replaced with crude glycerol as a carbon source. During this culture yeast produced 81.9 g L−1 of erythritol, which corresponded to a 0.9 g L−1 h−1 volumetric erythritol production rate and a yield of erythritol production of 0.4 g g−1. The fermentation broth was desalinated and decolorized by ion exclusion, ion exchange and sorption on activated carbon. The final solution contained only erythritol. The presented technology follows the vision of a circular economy by turning waste into products and using renewable materials as a feedstock. This study supports the potential of the industrially relevant wild type Yarrowia lipolytica strain for intensified and efficient erythritol production. The proposed technology is ecofriendly, requires a small number of purification steps and generates low amount of wastes. The final solution is rich in erythritol and can be applied directly as a sweet low-calorie food additive.

Erythritol production from raw materials by Yarrowia lipolytica

Erythritol production from raw materials by Yarrowia lipolytica

Improving Erythritol Production of Aureobasidium pullulans from Xylose by Mutagenesis and Medium Optimization.

The biotransformation of xylose, the second most abundant sugar, has been a hot topic in recent years. In this work, Aureobasidium pullulans CGMCC3.0837 was subjected to UV mutagenesis to improve its erythritol production from xylose. The erythritol production of the obtained mutant ER35 was 50.92% (17.28g/L) higher than that of the parent strain. Response surface methodology was applied to optimize the medium composition. Yeast extract, KH2PO4, and citric acid were the key factors influencing erythritol synthesis, and the optimal concentrations were 17.82, 0.76, and 6.60g/L, respectively. Under the optimized conditions, 31.75 and 31.42g/L erythritol were obtained in shake flasks and in a 5-L fermentor, respectively. ER35 also showed a good consuming ability on xylose mother liquor with a final erythritol production of 26.35g/L. This report provided insights into the potential of A. pullulans for the production of erythritol using xylose as a carbon source.

Erythritol production by engineered Yarrowia lipolytica

Medium-chain alcohols are used to produce solvents, surfactants, lubricants, waxes, creams, and cosmetics. In this study, we engineered the oleaginous yeast Yarrowia lipolytica to produce 1-decanol from glucose. Expression of a fatty acyl-CoA reductase from Arabidopsis thaliana in strains of Y. lipolytica previously engineered to produce medium-chain fatty acids resulted in the production of 1-decanol. However, the resulting titers were very low (<10 mg/mL), most likely due to product catabolism. In addition, these strains produced small quantities of 1-hexadecanol and 1-octadecanol. Deleting the major peroxisome assembly factor Pex10 was found to significantly increase 1-decanol production, resulting in titers exceeding 500 mg/L. It also increased 1-hexadecanoland and 1-octadecanol titers, though the resulting increases were less than those for 1-decanol. These results demonstrate that Y. lipolytica can potentially be used for the industrial production of 1-decanol and other fatty alcohols from simple sugars.

Optimization of a low-cost hyperosmotic medium and establishing the fermentation kinetics of erythritol production by Yarrowia lipolytica from crude glycerol

ABSTRACTThe production of erythritol by Yarrowia lipolytica from low-cost substitutable substrates for high yield was investigated. Crude glycerol, urea, and NaCl related to osmotic pressure were the most significant factors affecting erythritol production. An artificial neural network model and genetic algorithm were used to search the optimal composition of the significant factors and locate the resulting erythritol yield. Medium with 232.39 g/L crude glycerol, 1.57 g/L urea, and 31.03 g/L NaCl led to predictive maximum erythritol concentration of 110.7 g/L. The erythritol concentration improved from 50.4 g/L to 109.2 g/L with the optimized medium, which was reproducible. Erythritol fermentation kinetics were investigated in a batch system. Multistep fermentation kinetic models with hyperosmotic inhibitory effects were developed. The resulting mathematical equations provided a good description of temporal variations such as microbial growth (X), substrate consumption (S), and product formation (P) in erythritol fermentation. The accordingly derived model is the first reported model for fermentative erythritol production from glycerol, providing useful information to optimize the growth of Y. lipolytica and contributing visual description for the erythritol fermentation process under high osmotic pressure, as well as improvement of productivity and efficiency.

MAP Kinase Hog1 Regulates Metabolic Changes Induced by Hyperosmotic Stress.

Upon hyperosmotic stress, the High Osmolarity Glycerol (HOG) pathway is activated, resulting in phosphorylation of the stress-activated protein kinasei Hog1 (O'Rourke et al., 2002; Saito and Posas, 2012; Brewster and Gustin, 2014). The Hog1/mitogen-activated protein kinase-mediated signaling cascades are essential for sensing hyperosmotic stress and for transmitting these signals to the nucleus to modulate gene expression (O'Rourke et al., 2002; Saito and Posas, 2012; Brewster and Gustin, 2014). MAPK-mediated cascade pathways are composed of three serine/threonine protein kinases (MAPK kinase kinase, MAPK kinase, and MAPK). This signal transduction pathway is well-conserved in eukaryotes (O'Rourke et al., 2002; Saito and Posas, 2012; Brewster and Gustin, 2014). Phosphorylated Hog1 stimulates expression of genes encoding enzymes involved in glycerol production and uptake (O'Rourke et al., 2002; Saito and Posas, 2012; Gomar-Alba et al., 2013; Lee et al., 2013; Babazadeh et al., 2014; Brewster and Gustin, 2014). The glycerol production starts with the reduction of the glycolytic intermediate di-hydroxyl-acetone phosphate (DHAP) to glycerol-3-phosphate (G3P) catalyzed by the NAD+-dependent glycerol-3-phospate dehydrogenase (GPD; Ansell et al., 1997; Pahlman et al., 2001; Valadi et al., 2004). Increased expression of GPD1 enhances glycerol production under hyperosmotic stress (Albertyn et al., 1994; Rep et al., 1999). In addition, Hog1 appears to stimulate the 6-phosphofructo-2-kinase Pfk26, which produces fructose-2,6-diphosphate (F26DP), an allosteric activator of the glycolytic enzyme phosphofructokinase (Pfk1; Dihazi et al., 2004). Hog1 is rapidly phosphorylated upon curcumin treatment, an active polyphenol derived from the spice turmeric (Azad et al., 2014). Trichosporonoides oedocephalis is known to produce large amounts of polyols (erythritol and glycerol). Tohog1 null mutation increased erythritol production and decreased glycerol production, respectively (Li et al., 2016). Therefore, Hog1 is considered to have an important role in the metabolic control, however little is known regarding the changes of metabolites induced by Hog1 from a metabolic perspective. Nuclear magnetic resonance (NMR) spectroscopy is a rapid method that requires minimal sample preparation (Beckonert et al., 2007; Kim et al., 2010). In addition, NMR is a useful technique for structure elucidation due to its various two-dimensional measurements, which makes NMR an ideal tool for the identification and quantification of metabolites (Beckonert et al., 2007; Kim et al., 2010).

Genetic engineering of Synechocystis PCC6803 for the photoautotrophic production of the sweetener erythritol.

BackgroundErythritol is a polyol that is used in the food and beverage industry. Due to its non-caloric and non-cariogenic properties, the popularity of this sweetener is increasing. Large scale production of erythritol is currently based on conversion of glucose by selected fungi. In this study, we describe a biotechnological process to produce erythritol from light and CO2, using engineered Synechocystis sp. PCC6803.MethodsBy functionally expressing codon-optimized genes encoding the erythrose-4-phosphate phosphatase TM1254 and the erythrose reductase Gcy1p, or GLD1, this cyanobacterium can directly convert the Calvin cycle intermediate erythrose-4-phosphate into erythritol via a two-step process and release the polyol sugar in the extracellular medium. Further modifications targeted enzyme expression and pathway intermediates.ConclusionsAfter several optimization steps, the best strain, SEP024, produced up to 2.1 mM (256 mg/l) erythritol, excreted in the medium.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0458-y) contains supplementary material, which is available to authorized users.

Enhanced production of erythritol and mannitol by Yarrowia lipolytica in media containing surfactants.

Various chemical compounds, including surfactants, when introduced to culture media may increase the permeability of cellular membranes and thereby affect the quantity of metabolites excreted by cells. The aim of the present study was to evaluate the impact of detergents including Triton X-100, Span 20 and Tween 80 on erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 in a shake-flask experiment, batch and fed-batch cultures. When Span 20 was added to a fed-batch culture with glycerol as a carbon source (300 g L−1), erythritol production increased by 15% compared to the culture without the surfactant where it reached 142 g L−1 after 5 days, which corresponded to 0.47 g g−1 yield and productivity of 1.1 g L−1 h−1. Therefore, it was concluded that Span 20 considerably enhanced the production of this polyol from glycerol.

Construction of an Efficient Mutant Strain of Trichosporonoides oedocephalis with HOG1 Gene Deletion for Production of Erythritol.

The mitogen-activated protein kinase HOG1 (high-osmolarity glycerol response pathway) plays a crucial role in the response of yeast to hyperosmotic shock. Trichosporonoides oedocephalis produces large amounts of polyols (,e.g., erythritol and glycerol) in a culture medium. However, the effects of HOG1 gene knockout and environmental stress on the production of these polyols have not yet been studied. In this study, a To-HOG1 null mutation was constructed in T. oedocephalis using the loxP-Kan-loxP/Cre system as replacement of the targeted genes, and the resultant mutants showed much smaller colonies than the wild-type controls. Interestingly, compared with the wild-type strains, the results of shake-flask culture showed that To-HOG1 null mutation increased erythritol production by 1.44-fold while decreasing glycerol production by 71.23%. In addition, this study investigated the effects of citric acid stress on the T. oedocephalis HOG1 null mutants and the wild-type strain. When the supplementation of citric acid in the fermentation medium was controlled at 0.3% (w/v), the concentration of erythritol produced from the wild-type and To-HOG1 knockout mutant strains improved by 18.21% and 21.65%, respectively.

A two-stage fermentation process of erythritol production by yeast Y. lipolytica from molasses and glycerol

In this study, a two-stage fermentation process of erythritol production based on molasses and glycerol was investigated. During the first stage, the biomass of Yarrowia lipolytica was grown on medium containing sucrose as the sole carbon source. In the second stage, production of erythritol was initiated by glycerol addition. To use molasses as a substrate for erythritol synthesis, sucrose utilization was established by expressing the Saccharomyces cerevisiae SUC2 gene. In this study, cultivation of yeast Y. lipolytica could produce 52–114g/L of erythritol. The productivity was 0.58–1.04g/L/h, and yield was 0.26–0.57g/g; the final biomasses yield ranged 17–41g/L. This is the first report describing erythritol production via industrial raw molasses and glycerol by Y. lipolytica. This work uses genetically modified strains of Y. lipolytica as tool for the direct conversion of affordable raw industrial molasses and glycerol into the value-added erythritol product.

High production of erythritol from Candida sorbosivorans SSE-24 and its inhibitory effect on biofilm formation of Streptococcus mutans

Amongst different isolates screened for erythritol production, isolate no. SSE-24 was found to be the best erythritol producer and identified as Candida sorbosivorans SSE-24. Statistical optimization was used to determine the optimum level of the significant variables for maximum erythritol production. The interactive effects of glucose, inoculum level and yeast extract were determined to be significant. The optimum medium composition for erythritol production was 160g/L glucose, 12g/L yeast extract, 10% inoculum level and 0.35g/L FeSO4⋅7H2O. The production of erythritol was successfully scaled up to a 30L level, where 60.20g/L of erythritol was produced, with a yield of 0.38g/g. The fermentation broth was purified by activated charcoal followed by vacuum concentration, ion exchange chromatography and crystallization. Purity of erythritol was further determined by NMR. Significant inhibitory effect of erythritol on growth (>78%) and biofilm formation (40.2%) of Streptococcus mutans enhances the importance of this study.

Proteomic Analysis of Erythritol-Producing Yarrowia lipolytica from Glycerol in Response to Osmotic Pressure.

Osmotic pressure is a critical factor for erythritol production with osmophilic yeast. Protein expression patterns of an erythritol-producing yeast, Yarrowia lipolytica, were analyzed to identify differentially-expressed proteins in response to osmotic pressure. In order to analyze intracellular protein levels quantitatively, two-dimensional gel electrophoresis was performed to separate and visualize the differential expression of the intracellular proteins extracted from Y. lipolytica cultured under low (3.17 osmol/kg) and high (4.21 osmol/kg) osmotic pressures. Proteomic analyses allowed identification of 54 differentially-expressed proteins among the proteins distributed in the range of pI 3-10 and 14.4-97.4 kDa molecular mass between the osmotic stress conditions. Remarkably, the main proteins were involved in the pathway of energy, metabolism, cell rescue, and stress response. The expression of such enzymes related to protein and nucleotide biosynthesis was inhibited drastically, reflecting the growth arrest of Y. lipolytica under hyperosmotic stress. The improvement of erythritol production under high osmotic stress was due to the significant induction of a range of crucial enzymes related to polyols biosynthesis, such as transketolase and triosephosphate isomerase, and the osmotic stress responsive proteins like pyridoxine-4-dehydrogenase and the AKRs family. The polyols biosynthesis was really related to an osmotic response and a protection mechanism against hyperosmotic stress in Y. lipolytica. Additionally, the high osmotic stress could also induce other cell stress responses as with heat shock and oxidation stress responses, and these responsive proteins, such as the HSPs family, catalase T, and superoxide dismutase, also had drastically increased expression levels under hyperosmotic pressure.

Erythritol production by Moniliella megachiliensis using nonrefined glycerol waste as carbon source.

The number of naphtha plants is being reduced due to a worldwide shift in energy sources. Consequently, a shortage of chemical materials heavily dependent on naphtha-oil, especially C4 compounds such as butene and butane-diol, is an urgent issue in chemical manufacturing. Erythritol is a rare C4 compound produced by fermentation processes using glucose as the carbon source. Since erythritol is considerably more expensive than hydrocarbons derived from naphtha-oil, a reduction in its cost is critical. We found that Moniliella megachiliensis, a highly osmotolerant yeast strain, can utilize nonrefined glycerol waste derived from palm oil or beef tallow and convert it to erythritol. Cell growth on glycerol was almost the same as on glucose, and the cells could grow in up to 300mgml(-1) glycerol. When 200mgml(-1) nonrefined glycerol was supplied, the yield of erythritol from the glycerol was approx. 60%, which is slightly higher than that obtained using glucose. The cost of glycerol waste is considerably lower than that of glucose. Thus, the conversion of glycerol waste into valuable erythritol, proposed here, is attractive and promising from the viewpoint of ensuring a supply of C4 hydrocarbons and utilizing a waste natural resource. A shortage of C4 hydrocarbon depending much on naptha-oil has become urgent problem due to rapid reduction of naphtha plants together with global energy revolution. Erythritol, obtained by fermentation, is a rare C4 polyol that can be converted to C4 hydrocarbons. Erythritol is considerably expensive than hydrocarbons, a reduction in cost is critical issue. To meet this, we proposed to utilize low-cost glycerol waste from bio-diesel fuel as a carbon source. Moniliella megachiliensis successfully converted glycerol waste to erythritol. This proposal is promising to obtain C4 hydrocarbon substitute, and concomitantly to dispose a large amount of glycerol waste discharged.

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.

Optimization of Medium Composition for Erythritol Production from Glycerol by Yarrowia lipolytica Using Response Surface Methodology

Several factors affecting erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 strain were examined in batch fermentations. Ammonium sulfate, monopotassium phosphate, and sodium chloride were identified as critical medium components that determine the ratio of polyols produced. The central composite rotatable experimental design was used to optimize medium composition for erythritol production. The concentrations of ammonium sulfate, monopotassium phosphate, and sodium chloride in the optimized medium were 2.25, 0.22, and 26.4 g L−1, respectively. The C:N ratio was found as 81:1. In the optimized medium with 100 g L−1 of glycerol the Wratislavia K1 strain produced 46.9 g L−1 of erythritol, which corresponded to a 0.47 g g−1 yield and a productivity of 0.85 g L−1 hr−1. In the fed-batch mode and medium with the total concentration of glycerol at 300 g L−1 and C:N ratio at 81:1, 132 g L−1 of erythritol was produced with 0.44 g g−1 yield and a productivity of 1.01 g L−1 hr−1.