Purification of Natural Pigments Violacein and Deoxyviolacein Produced by Fermentation Using Yarrowia lipolytica.

Violacein and deoxyviolacein are bis-indole pigments synthesized by a number of microorganisms. The present study describes the biosynthesis of a mixture of violacein and deoxyviolacein using a genetically modified Y. lipolytica strain as a production chassis, the subsequent extraction of the intracellular pigments, and ultimately their purification using column chromatography. The results show that the optimal separation between the pigments occurs using an ethyl acetate/cyclohexane mixture with different ratios, first 65:35 until both pigments were clearly visible and distinguishable, then 40:60 to create a noticeable separation between them and recover the deoxyviolacein, and finally 80:20, which allows the recovery of the violacein. The purified pigments were then analyzed by thin-layer chromatography and nuclear magnetic resonance.

Increased Cordycepin Production in Yarrowia lipolytica Using Combinatorial Metabolic Engineering Strategies.

As the first nucleoside antibiotic discovered in fungi, cordycepin, with its various biological activities, has wide applications. At present, cordycepin is mainly obtained from the natural fruiting bodies of Cordyceps militaris. However, due to long production periods, low yields, and low extraction efficiency, harvesting cordycepin from natural C. militaris is not ideal, making it difficult to meet market demands. In this study, an engineered Yarrowia lipolytica YlCor-18 strain, constructed by combining metabolic engineering strategies, achieved efficient de novo cordycepin production from glucose. First, the cordycepin biosynthetic pathway derived from C. militaris was introduced into Y. lipolytica. Furthermore, metabolic engineering strategies including promoter, protein, adenosine triphosphate, and precursor engineering were combined to enhance the synthetic ability of engineered strains of cordycepin. Fermentation conditions were also optimized, after which, the production titer and yields of cordycepin in the engineered strain YlCor-18 under fed-batch fermentation were improved to 4362.54 mg/L and 213.85 mg/g, respectively, after 168 h. This study demonstrates the potential of Y. lipolytica as a cell factory for cordycepin synthesis, which will serve as the model for the green biomanufacturing of other nucleoside antibiotics using artificial cell factories.

Altering the fatty acid profile of Yarrowia lipolytica to mimic cocoa butter by genetic engineering of desaturases.

BACKGROUND: Demand for Cocoa butter is steadily increasing, but the supply of cocoa beans is naturally limited and under threat from global warming. One route to meeting the future demand for cocoa butter equivalent (CBE) could be to utilize microbial cell factories such as the oleaginous yeast Yarrowia lipolytica. RESULTS: The main goal was to achieve triacyl-glycerol (TAG) storage lipids in Y. lipolytica mimicking cocoa butter. This was accomplished by replacing the native Δ9 fatty acid desaturase (Ole1p) with homologs from other species and changing the expression of both Ole1p and the Δ12 fatty acid desaturase (Fad2p). We thereby abolished the palmitoleic acid and reduced the linoleic acid content in TAG, while the oleic acid content was reduced to approximately 40 percent of the total fatty acids. The proportion of fatty acids in TAG changed dramatically over time during growth, and the fatty acid composition of TAG, free fatty acids and phospholipids was found to be very different. CONCLUSIONS: We show that the fatty acid profile in the TAG of Y. lipolytica can be altered to mimic cocoa butter. We also demonstrate that a wide range of fatty acid profiles can be achieved while maintaining good growth and high lipid accumulation, which, together with the ability of Y. lipolytica to utilize a wide variety of carbon sources, opens up the path toward sustainable production of CBE and other food oils.

Studies on the Catalytic Properties of Crude Freeze-Dried Preparations of Yarrowia lipolytica Extracellular Lipases for Geranyl Ester Derivative Synthesis.

The study aimed to evaluate the impact of selected factors of the freeze-drying process on the hydrolytic and synthetic activity of the extracellular lipases of Y. lipolytica KKP 379 and to attempt the use of the crude enzyme preparation as a biocatalyst in the synthesis of geranyl 4-hydroxyphenylpropanoate. Antioxidant and antibacterial properties of the geranyl ester derivative were also investigated in order to evaluate their usefulness as a novel food additive. The studies confirmed that freeze-drying was an effective method of dehydrating yeast supernatant and allowed for obtaining lyophilizates with low water activity from 0.055 to 0.160. The type and concentration of the additive (2-6% whey protein hydrolyzate, 0.5% and 1% ammonium sulphate) had a significant effect on the hydrolytic activity of enzyme preparations, while the selected variants of drying temperature during the freeze-drying process were not significant (10 °C and 50 °C). Low yield of geranyl 4-hydroxyphenylopropionate was shown when the lyophilized supernatant was used (5.3%), but the yield of ester synthesis increased when the freeze-dried Y. lipolytica yeast biomass was applied (47.9%). The study confirmed the antioxidant properties of the synthesized ester by the DPPH• and CUPRAC methods, as well as higher antibacterial activity against tested bacteria than its precursor with 0.125 mM MIC (minimal inhibitory concentration) against L. monocytogenes.

Analysis of the Yarrowia lipolytica proteome reveals subtle variations in expression levels between lipogenic and non-lipogenic conditions.

Oleaginous yeasts have the ability to store greater than 20% of their mass as neutral lipids, in the form of triacylglycerides. The ATP citrate lyase is thought to play a key role in triacylglyceride synthesis, but the relationship between expression levels of this and other related enzymes is not well understood in the role of total lipid accumulation conferring the oleaginous phenotype. We conducted comparative proteomic analyses with the oleaginous yeast, Yarrowia lipolytica, grown in either nitrogen-sufficient rich media or nitrogen-limited minimal media. Total proteins extracted from cells collected during logarithmic and late stationary growth phases were analyzed by 1D liquid chromatography, followed by mass spectroscopy. The ATP citrate lyase enzyme was expressed at similar concentrations in both conditions, in both logarithmic and stationary phase, but many upstream and downstream enzymes showed drastically different expression levels. In non-lipogenic conditions, several pyruvate enzymes were expressed at higher concentration. These enzymes, especially the pyruvate decarboxylase and pyruvate dehydrogenase, may be regulating carbon flux away from central metabolism and reducing the amount of citrate being produced in the mitochondria. While crucial for the oleaginous phenotype, the constitutively expressed ATP citrate lyase appears to cleave citrate in response to carbon flux upstream from other enzymes creating the oleaginous phenotype.

Yarrowia lipolytica L-asparaginase inhibits the growth and migration of lung (A549) and breast (MCF7) cancer cells.

L-asparaginase is an enzyme capable of hydrolyzing the asparagine to aspartic acid and ammonia. L-asparaginase is widely used in the treatment of acute lymphoblastic leukemia (ALL) and other cancers. Here, for the first time, the effects of a novel yeast L-asparaginase from Yarrowia lipolytica were studied on human lung (A549) and breast cancer (MCF7) cell lines as the solid cancer cell lines in terms of cell growth and metastasis inhibition. Functional analysis showed the L-asparagine deprivation mediated anti-proliferation effects by apoptosis induction and changes in the expression of target genes involved in apoptosis and migration pathways. The qRT-PCR analysis showed the higher expression levels of pro-apoptosis genes, including Bax, P53, caspase 3, caspase 8, and down-regulation of Bcl-2 anti-apoptotic gene in treated cells. On the other hand, there was no increase in ROS production in the treated cells. However, L-asparaginase treatment was able to significantly induce autophagy activation in A549 cells. Besides, wound healing assay showed that L-asparaginase could considerably inhibit the migration of A549 and MCF7 cells. Taken together, our results suggested that Yarrowia lipolytica L-asparaginase might be considered for enzyme therapy against breast and lung cancers.

Mitochondrial complex I structure reveals ordered water molecules for catalysis and proton translocation.

Mitochondrial complex I powers ATP synthesis by oxidative phosphorylation, exploiting the energy from ubiquinone reduction by NADH to drive protons across the energy-transducing inner membrane. Recent cryo-EM analyses of mammalian and yeast complex I have revolutionized structural and mechanistic knowledge and defined structures in different functional states. Here, we describe a 2.7-Å-resolution structure of the 42-subunit complex I from the yeast Yarrowia lipolytica containing 275 structured water molecules. We identify a proton-relay pathway for ubiquinone reduction and water molecules that connect mechanistically crucial elements and constitute proton-translocation pathways through the membrane. By comparison with known structures, we deconvolute structural changes governing the mammalian 'deactive transition' (relevant to ischemia-reperfusion injury) and their effects on the ubiquinone-binding site and a connected cavity in ND1. Our structure thus provides important insights into catalysis by this enigmatic respiratory machine.

Dietary supplementation of marine yeast Yarrowia lipolytica modulates immune response in Litopenaeus vannamei.

The immunostimulatory potential of the marine yeast Yarrowia lipolytica (D1 and N6 strains) administered orally was evaluated in the white shrimp Litopenaeus vannamei. Yeasts and commercial glucans were mixed with a commercial feed to formulate diets with a 1.1% concentration of immunostimulants. The shrimp were fed daily for a period of 21 days. Weekly determinations were performed for immunological parameters in hemolymph, such as total hemocyte count (THC), lysozyme activity (LYZ), prophenoloxidase activity, antioxidant enzymatic activities (superoxide dismutase [SOD], catalase [CAT], and peroxidases), and bactericidal activity against Vibrio parahaemolyticus. Expression profiles of penaeidin (PEN), lysozyme (LYZ), and prophenoloxidase (proPO) immune genes were evaluated in hemocytes. In general, an increase in the immune parameters was observed in shrimp fed yeast diet compared to glucan and the control diets. Yarrowia lipolytica, especially strain N6, provided maximum immunostimulatory effects evidenced by the increase of immune parameters (THC, LYZ, SOD, CAT) and gene expression profile. In conclusion, this study demonstrated that Y. lipolytica had immunostimulatory effects and increased bactericidal activity in L. vannamei hemocytes against V. parahaemolyticus. These findings open the path for the potential application of Y. lipolytica-based immunostimulant for shrimp aquaculture.

Increased campesterol synthesis by improving lipid content in engineered Yarrowia lipolytica.

Sterols attract increasing attention due to their important bioactivities. The oleaginous yeast Yarrowia lipolytica has large lipid droplets, which provide storage for the accumulated steroid compounds. In this study, we have successfully constructed a campesterol biosynthetic pathway by modifying the synthetic pathway of ergosterol in Y. lipolytica with different capacity of lipid synthesis. The results showed that the maximal campesterol production was produced in the engineered strain YL-D+M-E-, as the optimal lipid content. Furthermore, we found that campesterol mainly exists in the lipid droplets. The campesterol production was further accumulated through the overexpression of two copies of dhcr7. Finally, the maximal campesterol production of 837 mg/L was obtained using a 5-L bioreactor in the engineered YL-D+D+M-E-, exhibiting a 3.7-fold increase compared with the initial strain YL-D+E-. Our results demonstrate that the proper promotion of lipid content plays an important role in campesterol biosynthesis in Y. lipolytica, and what we found provides an effective strategy for the production of hydrophobic compounds.Key Points• Campesterol was biosynthesized by deleting erg5 and introducing heterologous dhcr7.• Campesterol production elevated via promotion of lipid content.• Campesterol was mainly found in lipid droplets.• Promotion of lipid content is an effective strategy to produce hydrophobic compounds.

Engineering Yarrowia lipolytica for Enhanced Production of Arbutin.

Arbutin, a glycoside, is derived from the leaves of several plants, including wheat, pear, and bearberry plants, and has a significant role in the treatment of melanoma, cystitis, and cough. Here, we aimed to modify Yarrowia lipolytica to produce arbutin. To construct the arbutin synthetic pathway in Y. lipolytica, three genes (chorismate pyruvate-lyase (UbiC), 4-hydroxybenzoate 1-hydroxylase (MNX1), and hydroquinone glucosyltransferase (AS)) were codon-optimized and heterologously expressed. To maximize arbutin production, seven arbutin-biosynthesis molecular targets were overexpressed, and we found that the individual strengthening of DHS1 and DHS2 led to an 8.9- and 7.8-fold improvement in arbutin yield, respectively. Through optimization, a maximum arbutin titer of 8.6 ± 0.7 g/L was achieved using the finally engineered strain, po1f-At09. Overall, this is the first report of heterologous arbutin synthesis in Y. lipolytica at a high titer. Furthermore, this work opens a possibility for the overproduction of shikimate pathway derivatives in Y. lipolytica.

Impact of uranium exposure on marine yeast, Yarrowia lipolytica: Insights into the yeast strategies to withstand uranium stress.

A marine yeast, Yarrowia lipolytica, was evaluated for morphological, physiological and biochemical responses towards uranium (U) exposure at pH 7.5. The yeast revealed biphasic U binding - a rapid biosorption resulting in ∼35% U binding within 15-30 min followed by a slow biomineralization process, binding up to ∼45.5% U by 24 h on exposure to 50 µM of uranyl carbonate. Scanning electron microscopy coupled with Energy Dispersive X-ray spectroscopy analysis of 24 h U challenged cells revealed the deposition of uranyl precipitates due to biomineralization. The loss of intracellular structures together with surface and subcellular localization of uranyl precipitates in 24 h U exposed cells were visualized by transmission electron microscopy. Cells treated with 50 µM U exhibited membrane permeabilization which was higher at 200 µM U. Enhanced reactive oxygen species (ROS) accumulation and lipid peroxidation, transient RNA degradation and protein oxidation were observed in U exposed cells. High superoxide dismutase levels coupled with uranium binding and bioprecipitation possibly helped in counteracting U stress in 50 µM U treated cells. Resistance to U toxicity apparently developed under prolonged uranyl (50 µM) incubations. However, cells could not cope up with toxicity at 200 µM U due to impairment of resistance mechanisms.

Quantitative Structure-Activity Relationship Model to Predict Antioxidant Effects of the Peptide Fraction Extracted from a Co-Culture System of Chlorella pyrenoidosa and Yarrowia lipolytica.

In this study, the antioxidant components in co-culture of Chlorella pyrenoidosa and Yarrowia lipolytica (3:1 ratio) were confirmed as trypsin-hydrolyzed peptides (EHPs). The EHPs were composed of 836 different peptides with molecular weights ranging from 639 to 3531 Da and were mainly composed of hydrophobic amino acids (48.1%). These peptides showed remarkable protective effects against oxidative stress in HepG2, which may be attributed to their structures. Furthermore, the mRNA and protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) were significantly lower in the peptide-treated group than in the control group, suggesting that the antioxidant enzyme-coding genes were not activated. The EC50 value of three peptides in the EHPs were in the order of AGYSPIGFVR (0.04 ± 0.002 mg/mL) > VLDELTLAR (0.09 ± 0.001 mg/mL) > LFDPVYLFDQG (0.41 ± 0.03 mg/mL); these results agreed with the prediction of the model (R2 > 0.9, Q2 > 0.5). Thus, EHPs show potential as potent new antioxidant agents.

Yarrowia lipolytica: more than an oleaginous workhorse.

Microbial production of fuels and chemicals offers a means by which sustainable product manufacture can be achieved. In this regard, Yarrowia lipolytica is a unique microorganism suitable for a diverse array of biotechnological applications. As a robust oleaginous yeast, it has been well studied for production of fuels and chemicals derived from fatty acids. However, thanks in part to newfound genetic tools and metabolic understanding, Y. lipolytica has been explored for high-level production of a variety of non-lipid products. This mini-review will discuss some of the recent research surrounding the ability of Y. lipolytica to support bio-based chemical production outside the realm of fatty acid metabolism including polyketides, terpenes, carotenoids, pentose phosphate-derived products, polymers, and nanoparticles.

An efficient method of Yarrowia lipolytica immobilization using oil- and emulsion-modified bacterial cellulose carriers

Background: Yarrowia lipolytica is a nonconventional, dimorphic yeast with multiple biotechnological applications. Considering the size of Y. lipolytica cells and a plethora of its morphological forms (spherical cells or hyphae and pseudohyphae), it is highly difficult to select a suitable carrier for this useful microorganism. Bacterial cellulose (BC) is currently considered one of the most promising immobilization carriers. In the current study, the usefulness of oil- and emulsion-modified BCs as a carrier for Y. lipolytica immobilization was investigated. Static and agitated cultures were conducted in media supplemented with oil or emulsion to improve carrier porosity. Results: It was found that the application of oil- and emulsion-modified BCs correlated with significantly higher efficiency of Y. lipolytica immobilization and hence higher yield than the yield achieved with an unmodified carrier. Increased efficiency of immobilization correlated with BC porosity-related parameters, which, in turn, depended on the size of oil droplets introduced into the culture medium. Moreover, changes in porosity-related parameters caused by the addition of oil or emulsion to the medium were observed when the cultures were conducted only under static conditions and not under agitated conditions. Conclusion: The application of oil- and emulsion-modified BCs as carriers significantly increased the efficiency of Y. lipolytica immobilization as compared to unmodified BC. The addition of oil or emulsion to the culture medium can be a simple but effective method to modify the porosity of BC-based carriers.

Rhodococcus and Yarrowia-Based Lipid Production Using Lignin-Containing Industrial Residues.

Improvement in biorefining technologies coupled with development of novel fermentation strategies and analysis will be paramount in establishing supplementary and sustainable biofuel pathways. Oleaginous microorganisms that are capable of accumulating triacylglycerides (TAGs) and fatty acid methyl esters (FAMEs), such as Rhodococcus and Yarrowia species, can be used to produce second-generation biofuels from non-food competing carbon sources. These "microbiorefineries" provide a pathway to upgrade agricultural and industrial waste streams to fungible fuels or precursors to chemicals and materials. Here we provide a general overview on cultivating Rhodococcus and Yarrowia on agro-waste/industrial biomass pretreatment waste streams to produce single-cell oils/lipids and preparing samples for FAME detection.

Functional assessment of hydrophilic domains of late embryogenesis abundant proteins from distant organisms.

Late embryogenesis abundant (LEA) proteins play a protective role during desiccation and oxidation stresses. LEA3 proteins are a major group characterized by a hydrophilic domain (HD) with a highly conserved repeating 11-amino acid motif. We compared four different HD orthologs from distant organisms: (i) DrHD from the extremophilic bacterium Deinococcus radiodurans; (ii) CeHD from the nematode Caenorhabditis elegans; (iii) YlHD from the yeast Yarrowia lipolytica; and (iv) BnHD from the plant Brassica napus. Circular dichroism spectroscopy showed that all four HDs were intrinsically disordered in phosphate buffer and then folded into α-helical structures with the addition of glycerol or trifluoroethanol. Heterologous HD expression conferred enhanced desiccation and oxidation tolerance to Escherichia coli. These four HDs protected the enzymatic activities of lactate dehydrogenase (LDH) by preventing its aggregation under desiccation stress. The HDs also interacted with LDH, which was intensified by the addition of hydrogen peroxide (H2 O2 ), suggesting a protective role in a chaperone-like manner. Based on these results, the HDs of LEA3 proteins show promise as protectants for desiccation and oxidation stresses, especially DrHD, which is a potential ideal stress-response element that can be applied in synthetic biology due to its extraordinary protection and stress resistance ability.

On cross-correlations, averages and noise in electron microscopy.

Biological samples are radiation-sensitive and require imaging under low-dose conditions to minimize damage. As a result, images contain a high level of noise and exhibit signal-to-noise ratios that are typically significantly smaller than 1. Averaging techniques, either implicit or explicit, are used to overcome the limitations imposed by the high level of noise. Averaging of 2D images showing the same molecule in the same orientation results in highly significant projections. A high-resolution structure can be obtained by combining the information from many single-particle images to determine a 3D structure. Similarly, averaging of multiple copies of macromolecular assembly subvolumes extracted from tomographic reconstructions can lead to a virtually noise-free high-resolution structure. Cross-correlation methods are often used in the alignment and classification steps of averaging processes for both 2D images and 3D volumes. However, the high noise level can bias alignment and certain classification results. While other approaches may be implicitly affected, sensitivity to noise is most apparent in multireference alignments, 3D reference-based projection alignments and projection-based volume alignments. Here, the influence of the image signal-to-noise ratio on the value of the cross-correlation coefficient is analyzed and a method for compensating for this effect is provided.

Production and structural modeling of a novel asparaginase in Yarrowia lipolytica.

Asparaginase catalyzes the conversion of asparagine into aspartic acid and ammonia. The enzyme has various industrial applications and it is considered as an anticancer drug for treatment of certain leukemias. In the current study, production of asparaginase was investigated by Yarrowia lipolytica as well as optimized its production and determined its molecular characteristics by in silico analysis. Y. lipolytica DSM3286 produced 17.14 U/ml of asparaginase in flask culture. Optimization of asparaginase production was done by response surface methodology and the enzyme production increases up to 102.85 U/ml. The enzyme production reached 210 U/ml in a bioreactor which is 12-fold more than flask culture containing non-optimized medium. Asparaginase gene of Y. lipolytica was identified and isolated on the basis of comparison with asparaginase gene sequences of other microorganisms. The gene has 981 nucleotides and its protein has 326 amino acids. According to in silico analysis, the secondary structure of the enzyme is composed of 9 α-helixes and 11 ß-sheets. Y. lipolytica produces type II asparaginase with high affinity for asparagine which is a suitable eukaryotic asparaginase for treatment of hematopoietic cancers. Hence, Y. lipolytica could be recommended as a new eukaryotic microbial source for the production of this important therapeutic enzyme.

Fermentation Conditions and Media Optimization for Isocitric Acid Production from Ethanol by Yarrowia lipolytica.

Isocitric acid exists in the form of four stereoisomers, of which only the threo-Ds-form (ICA) is a natural active compound, an intermediate of Krebs cycle, and suitable for nutritional and pharmaceutical use. In this paper, we propose a method for ICA production from ethanol by yeast Yarrowia lipolytica. The effects of temperature, pH of the medium, and aeration on the growth of the producer Y. lipolytica VKM Y-2373 and synthesis of ICA were studied. An optimal fermentation regime, which ensures a good growth of the producer and directed synthesis of the target product, was determined. The producer is advised to carry out cultivation at 29°C and various pH of the medium and the oxygen concentration (pH 5 and pO2 20-25% (of saturation) during the growth period and pH 6 and pO2 50-55% (of saturation) during the acid formation) on a nutrient medium containing an increased content of zinc (0.6 mg/L), iron (1.2 mg/L), and 30 mM itaconic acid (inhibitor of isocitrate lyase-the key enzyme of ICA metabolism) should also be introduced into the nutrition medium. Such fermentation production mode provides 90.5 g/L ICA with process selectivity of 80%, mass yield (YICA) of 0.77 g/g, and energy yield (ηICA) of 0.278 g/g.