Inhibition of hyphal formation together with biochar addition promotes erythritol production by Yarrowia lipolytica.

This study aimed to investigate the effect of hyphal formation in Yarrowia lipolytica and biochar addition on erythritol production by submerged fermentation. Hyphal formation significantly inhibited erythritol production by Y. lipolytica. Transcriptome analysis suggested that the impaired erythritol synthesis of hyphal cells was associated with the differential expression of genes involved in amino acid metabolism, lipid metabolism, and cell wall stability. Deletion of RAS2 responsible for yeast-to-hypha transition and EYD1 included in erythritol degradation blocked hyphal formation and improved erythritol production. Biochar prepared from corncob, sugarcane bagasse (SB), corn straw, peanut shell, coconut shell, and walnut shell (WS) had a positive effect on erythritol production, of which WS pyrolyzed at 500°C (WSc) performed the best in flask fermentation. In a 3.7 L bioreactor, 220.20 ± 10 g/L erythritol with a productivity of 2.30 ± 0.10 g/L/h was obtained in the presence of 1.4% (w/v) WSc and 0.7% SBc (SB pyrolyzed at 500°C) within 96 h. These results suggest that inhibition of hyphal formation together with biochar addition is an efficient way to promote erythritol production.

The gibberellic acid derived from the plastidial MEP pathway is involved in the accumulation of Bamboo mosaic virus.

A gene upregulated in Nicotiana benthamiana after Bamboo mosaic virus (BaMV) infection was revealed as 1-deoxy-d-xylulose-5-phosphate reductoisomerase (NbDXR). DXR is the key enzyme in the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway that catalyzes the conversion of 1-deoxy-d-xylulose 5-phosphate to 2-C-methyl-d-erythritol-4-phosphate. Knockdown and overexpression of NbDXR followed by BaMV inoculation revealed that NbDXR is involved in BaMV accumulation. Treating leaves with fosmidomycin, an inhibitor of DXR function, reduced BaMV accumulation. Subcellular localization confirmed that DXR is a chloroplast-localized protein by confocal microscopy. Furthermore, knockdown of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase, one of the enzymes in the MEP pathway, also reduced BaMV accumulation. The accumulation of BaMV increased significantly in protoplasts treated with isopentenyl pyrophosphate. Thus, the metabolites of the MEP pathway could be involved in BaMV infection. To identify the critical components involved in BaMV accumulation, we knocked down the crucial enzyme of isoprenoid synthesis, NbGGPPS11 or NbGGPPS2. Only NbGGPPS2 was involved in BaMV infection. The geranylgeranyl pyrophosphate (GGPP) synthesized by NbGGPPS2 is known for gibberellin synthesis. We confirmed this result by supplying gibberellic acid exogenously on leaves, which increased BaMV accumulation. The de novo synthesis of gibberellic acid could assist BaMV accumulation.

The Overexpression of YALI0B07117g Results in Enhanced Erythritol Synthesis from Glycerol by the Yeast Yarrowia lipolytica.

The unconventional yeast Yarrowia lipolytica is used to produce erythritol from glycerol. In this study, the role of the erythrose reductase (ER) homolog YALI0B07117g in erythritol synthesis was analyzed. The deletion of the gene resulted in an increased production of mannitol (308%) and arabitol (204%) before the utilization of these polyols began. The strain overexpressing the YALI0B07117g gene was used to increase the erythritol yield from glycerol as a sole carbon source in batch cultures, resulting in a yield of 0.4 g/g. The specific consumption rate (qs) increased from 5.83 g/g/L for the WT strain to 8.49 g/g/L for the modified strain and the productivity of erythritol increased from 0.28 g/(L h) for the A101 strain to 0.41 g/(L h) for the modified strain. The application of the research may prove positive for shortening the cultivation time due to the increased rate of consumption of the substrate combined with the increased parameters of erythritol synthesis.

Conferring thermotolerant phenotype to wild-type Yarrowia lipolytica improves cell growth and erythritol production.

In microbial engineering, heat stress is an important environmental factor modulating cell growth, metabolic flux distribution and the synthesis of target products. Yarrowia lipolytica, as a GARS (generally recognized as safe) nonconventional yeast, has been widely used in the food industry, especially as the host of erythritol production. Biomanufacturing economics is limited by the high operational cost of cooling energy in large-scale fermentation. It is of great significance to select thermotolerant Y. lipolytica to reduce the cooling cost and elucidate the heat-resistant mechanism at molecular level. For this purpose, we performed adaptive evolution and obtained a thermotolerant strain named Y. lipolytica BBE-18. Transcriptome analysis allows us to identify four genes in thiamine metabolism pathway that are responsible for the complicated thermotolerant phenotype. The heat-resistant phenotype was validated with the model strain Y. lipolytica Po1f by overexpression of single and combined genes. Then, conferring the thermotolerant phenotype to the wild-type Y. lipolytica BBE-17 enable the strain to produce three-times more erythritol of the control strain with 3°C higher than optimal cultivation temperature. To our knowledge, this is the first report on engineering heat-resistant phenotype to improve the erythritol production in Y. lipolytica. However, due to the increase of culture temperature, a large amount of adenosine triphosphate is consumed to ensure the life activities of Y. lipolytica which limits the potential of cell synthetic products to a certain extent. Even so, this study provides a reference for Y. lipolytica to produce other products under high temperature.

Multiple gene integration to promote erythritol production on glycerol in Yarrowia lipolytica.

OBJECTIVE: Erythritol (1,2,3,4-butanetetrol) is a 4-carbon sugar alcohol that occurs in nature as a metabolite or storage compound. In this study, a multiple gene integration strategy was employed to enhance erythritol production in Y. lipolytica. RESULTS: The effects on the production of erythritol in Y. lipolytica of seven key genes involved in the erythritol synthesis pathway were evaluated individually, among which transketolase (TKL1) and transaldolase (TAL1) showed important roles in enhancing erythritol production. The combined overexpression of four genes (GUT1, TPI1, TKL1, TAL1) and disruption of the EYD1 gene (encoding erythritol dehydrogenase), resulted in produce approximately 40 g/L erythritol production from glycerol. Further enhanced erythritol synthesis was obtained by overexpressing the RKI1 gene (encoding ribose 5-phosphate isomerase) and the AMPD gene (encoding AMP deaminase), indicating for the first time that these two genes are also related to the enhancement of erythritol production in Y. lipolytica. CONCLUSIONS: A combined gene overexpression strategy was developed to efficiently improve the production of erythritol in Y. lipolytica, suggesting a great capacity and promising potential of this non-conventional yeast in converting glycerol into erythritol.

Recent advances in the biotechnological production of erythritol and mannitol.

Dietary habits that include an excess of added sugars have been strongly associated with an increased risk of obesity, heart disease, diabetes, and tooth decay. With this association in view, modern food systems aim to replace added sugars with low calorie sweeteners, such as polyols. Polyols are generally not carcinogenic and do not trigger a glycemic response. Furthermore, owing to the absence of the carbonyl group, they are more stable compared to monosaccharides and do not participate in Maillard reactions. As such, since polyols are stable at high temperatures, and they do not brown or caramelize when heated. Therefore, polyols are widely used in the diets of hypocaloric and diabetic patients, as well as other specific cases where controlled caloric intake is required. In recent years, erythritol and mannitol have gained increased importance, especially in the food and pharmaceutical industries. In these areas, research efforts have been made to improve the productivity and yield of the two polyols, relying on biotechnological manufacturing methods. The present review highlights the recent advances in the biotechnological production of erythritol and mannitol and summarizes the benefits of using the two polyols in the food and pharmaceutical industries.

Combining genetically-encoded biosensors with high throughput strain screening to maximize erythritol production in Yarrowia lipolytica.

Erythritol is an important sweetener ingredient and chemical precursor for synthesizing materials with phase transition behavior. Commercial erythritol is primarily produced by industrial fermentation. Further strain engineering necessitates the development of high throughput screening method for rapid detection and screening of mutant strain libraries. In this work, we took advantage of the erythritol-responsive transcription factor EryD, and constructed a sensor-regulator system for rapid screening and characterization of erythritol overproducers. We configured the optimal architecture of the EryD sensor-regulator construct with improved sensitivity, specificity and dynamic response range. Coupled with mutagenesis and strain screening based on biosensors, we rapidly screened and characterized a strain library containing 1152 mutants derived from combined UV and ARTP mutagenesis, in a relatively short period of time (1 week). The optimal strain produced more than 148 g/L erythritol in bench-top reactors. This work provides a reference for other metabolic engineering researchers to develop industrially-relevant strains. The reported framework enables us to rapidly improve strain performance and engineer efficient microbial cell factories for industrial applications.

A review of polyols - biotechnological production, food applications, regulation, labeling and health effects.

Food research is constantly searching for new ways to replace sugar. This is due to the negative connotations of sugar consumption on health which has driven consumer demand for healthier products and is reflected on a national level by the taxation of sugary beverages. Sugar alcohols, a class of polyols, are present in varying levels in many fruits and vegetables and are also added to foods as low calorific sweeteners. The most commonly used polyols in food include sorbitol, mannitol, xylitol, erythritol, maltitol, lactitol and isomalt. Of these, microorganisms can produce sorbitol, mannitol, xylitol and erythritol either naturally or through genetic engineering. Production of polyols by microbes has been the focus of a lot of research for its potential as an alternative to current industrial scale production by chemical synthesis but can also be used for in situ production of natural sweeteners in fermented products using microbes approved for use in foods. This review on the generation of these natural sweetening compounds by microorganisms examines the current understanding and methods of microbial production of polyols that are applicable in the food industry. The review also considers the health benefits and effects of polyol usage and discusses regulations which are applicable to polyol use.

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

BACKGROUND: Yarrowia 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. RESULTS: Under 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. CONCLUSIONS: This 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.

De novo transcriptome of Gymnema sylvestre identified putative lncRNA and genes regulating terpenoid biosynthesis pathway.

Gymnema sylvestre is a highly valuable medicinal plant in traditional Indian system of medicine and used in many polyherbal formulations especially in treating diabetes. However, the lack of genomic resources has impeded its research at molecular level. The present study investigated functional gene profile of G. sylvestre via RNA sequencing technology. The de novo assembly of 88.9 million high quality reads yielded 23,126 unigenes, of which 18116 were annotated against databases such as NCBI nr database, gene ontology (GO), KEGG, Pfam, CDD, PlantTFcat, UniProt & GreeNC. Total 808 unigenes mapped to 78 different Transcription Factor families, whereas 39 unigenes assigned to CYP450 and 111 unigenes coding for enzymes involved in the biosynthesis of terpenoids including transcripts for synthesis of important compounds like Vitamin E, beta-amyrin and squalene. Among them, presence of six important enzyme coding transcripts were validated using qRT-PCR, which showed high expression of enzymes involved in methyl-erythritol phosphate (MEP) pathway. This study also revealed 1428 simple sequence repeats (SSRs), which may aid in molecular breeding studies. Besides this, 8 putative long non-coding RNAs (lncRNAs) were predicted from un-annotated sequences, which may hold key role in regulation of essential biological processes in G. sylvestre. The study provides an opportunity for future functional genomic studies and to uncover functions of the lncRNAs in G. sylvestre.

Antibacterial activity of 2-amino-4-hydroxypyrimidine-5-carboxylates and binding to Burkholderia pseudomallei 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthase.

Enzymes in the methylerythritol phosphate pathway make attractive targets for antibacterial activity due to their importance in isoprenoid biosynthesis and the absence of the pathway in mammals. The fifth enzyme in the pathway, 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), contains a catalytically important zinc ion in the active site. A series of de novo designed compounds containing a zinc binding group was synthesized and evaluated for antibacterial activity and interaction with IspF from Burkholderia pseudomallei, the causative agent of Whitmore's disease. The series demonstrated antibacterial activity as well as protein stabilization in fluorescence-based thermal shift assays. Finally, the binding of one compound to Burkholderia pseudomallei IspF was evaluated through group epitope mapping by saturation transfer difference NMR.

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.

A novel osmotic pressure strategy to improve erythritol production by Yarrowia lipolytica from glycerol.

Polyethylene glycol (PEG) is polymer that was used to replace NaCl (reference media) as an osmotic stress agent for the synthesis of erythritol by the osmophilic yeast Yarrowia lipolytica. Two strains, the wild-type strain IMUFRJ 50682 and the lab strain W29, were grown in the presence of PEG of different molecular weights. For strain IMUFRJ 50682, the erythritol titer was increased by 40% in the presence of PEG2000 as compared to the reference media (with NaCl). A similar increase was also observed for strain W29, except that it occurred in the presence of PEG6000. Moreover, in those experimental conditions neither strain produced mannitol, in contrast to the control medium. These results highlight that PEG could be used to increase erythritol productivity and to simultaneously inhibit mannitol synthesis, representing a good substitute for NaCl as an osmotic stress agent.

Erythritol production by yeasts: a snapshot of current knowledge.

Erythritol is a four-carbon sugar alcohol produced by microorganisms as an osmoprotectant. It could be used as a natural sweetener in the pharmaceutical and food industries. Here, a snapshot of current knowledge on erythritol metabolism and synthesis, optimization of its production and more precise process and producer strain improvement is presented.

Selective production of deacetylated mannosylerythritol lipid, MEL-D, by acetyltransferase disruption mutant of Pseudozyma hubeiensis.

Mannosylerythritol lipids (MELs) are produced by several smut fungi of the Ustilaginaceae family; they are promising microbial biosurfactants and have excellent surface-active and self-assembling properties. Pseudozyma hubeiensis is a candidate for abundant MEL production and produces large amounts of 4-O-[(4'-mono-O-acetyl-2',3'-di-O-alkanoyl)-ß-d-mannopyranosyl]-meso-erythritol (MEL-C). An acetyltransferase disruption mutant of P. hubeiensis, SY62-MM36, was obtained to selectively produce deacetylated 4-O-[(2',3'-di-O-alkanoyl)-ß-d-mannopyranosyl]-meso-erythritol (MEL-D), and the structures of the products were determined. Lower mobility of major spots of the mutant on silica gel thin-layer chromatography verified its more hydrophilic nature than that of wild-type MEL-A, B, and C. Structural analyses confirmed the product to be MEL-D, which comprises acyl chains of caproic acid (C6:0), capric acid (C10:0), and lauric acid (C12:0). The critical micelle concentration (CMC) and the surface tension (γCMC) of the MEL-D were 2.0 × 10-5 M and 29.7 mN/m, respectively. SY62-MM36 also produced a minor product that was estimated as triacylated MEL-D. The triacylated MEL-D had a CMC of 3.5 × 10-5 M and a γCMC of 29.6 mN/m. In water, MEL-D formed a lamella liquid crystal phase over a broad range of concentrations. By fed-batch cultivation, the mutant produced 91.6 ± 6.3 g/L of MEL-D for 7 days.

Recent advances in biological production of erythritol.

Erythritol is a natural sweetener commonly used in the food and pharmaceutical industries. Produced by microorganisms as an osmoprotectant, it is an ideal sucrose substitute for diabetics or overweight persons due to its almost zero calorie content. Currently, erythritol is produced on an industrial scale through the fermentation of sugars by some yeasts, such as Moniliella sp. However, the popularity of erythritol as a sweetener is still small because of its high retail price. This creates an opportunity for further process improvement. Recent years have brought the rapid development of erythritol biosynthesis methods from the low-cost substrates, and a better understanding of the metabolic pathways leading to erythritol synthesis. The yeast Yarrowia lipolytica emerges as an organism effectively producing erythritol from pure or crude glycerol. Moreover, novel erythritol producing organisms and substrates may be taken into considerations due to metabolic engineering. This review focuses on the modification of erythritol production to use low-cost substrates and metabolic engineering of the microorganisms in order to improve yield and productivity.

Erythritol as sweetener-wherefrom and whereto?

Erythritol is a naturally abundant sweetener gaining more and more importance especially within the food industry. It is widely used as sweetener in calorie-reduced food, candies, or bakery products. In research focusing on sugar alternatives, erythritol is a key issue due to its, compared to other polyols, challenging production. It cannot be chemically synthesized in a commercially worthwhile way resulting in a switch to biotechnological production. In this area, research efforts have been made to improve concentration, productivity, and yield. This mini review will give an overview on the attempts to improve erythritol production as well as their development over time.

Characterization of erythrose reductase from Yarrowia lipolytica and its influence on erythritol synthesis.

BACKGROUND: Erythritol is a natural sweetener that is used in the food industry. It is produced as an osmoprotectant by bacteria and yeast. Due to its chemical properties, it does not change the insulin level in the blood, and therefore it can be safely used by diabetics. Previously, it has been shown that erythrose reductase (ER), which catalyzes the final step, plays a crucial role in erythritol synthesis. ER reduces erythrose to erythritol with NAD(P)H as a cofactor. Despite many studies on erythritol synthesis by Yarrowia lipolytica, the enzymes involved in this metabolic pathway have ever been described. RESULTS: The gene YALI0F18590g encoding the predicted erythrose reductase from Y. lipolytica was overexpressed, and its influence on erythritol synthesis was studied. The amino acid sequence of the Y. lipolytica ER showed a high degree of similarity to the previously described erythrose reductases from known erythritol producers, such as Candida magnoliae and Moniliella megachiliensis. Here, we found that the gene overexpression results in an enhanced titer of erythritol of 44.44 g/L (20% over the control), a yield of 0.44 g/g and productivity of 0.77 g/L/h. Moreover, on purification and characterization of the enzyme we found that it displays the highest activity at 37 °C and pH 3.0. The effects of various metal ions (Zn2+, Cu2+, Mn2+, Fe2+) on erythrose reductase were investigated. The addition of Zn2+ ions at 0.25 mM had a positive effect on the activity of erythrose reductase from Y. lipolytica, as well as on the erythritol production. CONCLUSIONS: In this study we identified, overexpressed and characterized a native erythrose reductase in Y. lipolytica. Further optimizations of this strain via metabolic pathway engineering and media optimization strategies enabled 54 g/L to be produced in a shake-flask experiment. To date, this is the first reported study employing metabolic engineering of the native gene involved in the erythritol pathway to result in a high titer of the polyol. Moreover, it indicates the importance of environmental conditions for genetic targets in metabolic engineering.

Identification and characterization of EYK1, a key gene for erythritol catabolism in Yarrowia lipolytica.

Erythritol is a four-carbon sugar alcohol synthesized by osmophilic yeasts, such as Yarrowia lipolytica, in response to osmotic stress. This metabolite has application as food additive due to its sweetening properties. Although Y. lipolytica can produce erythritol at a high level from glycerol, it is also able to consume it as carbon source. This ability negatively affects erythritol productivity and represents a serious drawback for the development of an efficient erythritol production process. In this study, we have isolated by insertion mutagenesis a Y. lipolytica mutant unable to grow on erythritol. Genomic characterization of the latter highlighted that the mutant phenotype is directly related to the disruption of the YALI0F01606g gene. Several experimental evidences suggested that the identified gene, renamed EYK1, encodes an erythrulose kinase. The mutant strain showed an enhanced capacity to produce erythritol as compared to the wild-type strain. Moreover, in specific experimental conditions, it is also able to convert erythritol to erythrulose, another compound of biotechnological interest.

Enhancing erythritol productivity in Yarrowia lipolytica using metabolic engineering.

Erythritol (1,2,3,4-butanetetrol) is a four-carbon sugar alcohol with sweetening properties that is used by the agrofood industry as a food additive. In this study, we demonstrated that metabolic engineering can be used to improve the production of erythritol from glycerol in the yeast Yarrowia lipolytica. The best results were obtained using a mutant that overexpressed GUT1 and TKL1, which encode a glycerol kinase and a transketolase, respectively, and in which EYK1, which encodes erythrulose kinase, was disrupted; the latter enzyme is involved in an early step of erythritol catabolism. In this strain, erythritol productivity was 75% higher than in the wild type; furthermore, the culturing time needed to achieve maximum concentration was reduced by 40%. An additional advantage is that the strain was unable to consume the erythritol it had created, further increasing the process's efficiency. The erythritol productivity values we obtained here are among the highest reported thus far.