Application of freeze-dried Yarrowia lipolytica biomass in the synthesis of lipophilic antioxidants.

OBJECTIVE: The aim of the study was to evaluate the possibility of using Y. lipolytica biomass as a whole-cell catalyst in the synthesis of lipophilic antioxidants, with the example of esterification of five phenolic acids with 1-butanol. RESULTS: Freeze-dried Y. lipolytica biomass was successfully applied as a biocatalyst in the synthesis of esters of phenylpropanoic acid derivatives with 75-98% conversion. However, in the case of phenylacetic acid derivatives, results below 10% were obtained. The biological activity of phenolic acid esters was strongly associated with their chemical structures. Butyl 3-(4-hydroxyphenyl)propanoate showed an IC50 value of 19 mg/ml (95 mM) and TEAC value of 0.427. Among the compounds tested, butyl esters of 3-(4-hydroxyphenyl)propanoic and 4-hydroxyphenylacetic acids exhibited the highest antifungal activity. CONCLUSIONS: Lipophilization of phenolic acids achieved by enzymatic esterification creates prospects for using these compounds as food additives with antioxidant properties in lipid-rich food matrices.

Application of a New Engineered Strain of Yarrowia lipolytica for Effective Production of Calcium Ketoglutarate Dietary Supplements.

The present study aimed to develop a technology for the production of dietary supplements based on yeast biomass and α-ketoglutaric acid (KGA), produced by a new transformant of Yarrowia lipolytica with improved KGA biosynthesis ability, as well to verify the usefulness of the obtained products for food and feed purposes. Transformants of Y. lipolytica were constructed to overexpress genes encoding glycerol kinase, methylcitrate synthase and mitochondrial organic acid transporter. The strains were compared in terms of growth ability in glycerol- and oil-based media as well as their suitability for KGA biosynthesis in mixed glycerol-oil medium. The impact of different C:N:P ratios on KGA production by selected strain was also evaluated. Application of the strain that overexpressed all three genes in the culture with a C:N:P ratio of 87:5:1 allowed us to obtain 53.1 g/L of KGA with productivity of 0.35 g/Lh and yield of 0.53 g/g. Finally, the possibility of obtaining three different products with desired nutritional and health-beneficial characteristics was demonstrated: (1) calcium α-ketoglutarate (CaKGA) with purity of 89.9% obtained by precipitation of KGA with CaCO3, (2) yeast biomass with very good nutritional properties, (3) fixed biomass-CaKGA preparation containing 87.2 µg/g of kynurenic acid, which increases the health-promoting value of the product.

Characterization of the promoter, downstream target genes and recognition DNA sequence of Mhy1, a key filamentation-promoting transcription factor in the dimorphic yeast Yarrowia lipolytica.

Msn2/Msn4-family zinc finger transcription factors play important roles in stress response in yeast. However, some members of this family show significant functional divergence in different species. Here, we report that in the dimorphic yeast Yarrowia lipolytica, the Msn2/Msn4-like protein Mhy1 is a key regulator of yeast-to-hypha dimorphic transition but not stress response. Both MHY1 deletion and overexpression affect filamentation. In contrast, YlMsn4, the other Msn2/Msn4-like protein, regulates tolerance to acid-induced stress. We show that MHY1 has an unusually long (about 3800 bp) promoter featuring an upstream located enhancer and a double stress response element (STRE) motif, the latter of which mediates Mhy1's regulation on its own transcription. Transcriptome profiling conducted in wild-type strain, mhy1Δ mutant and MHY1-overexpressing mutant revealed about 100 genes that are highly differentially expressed (≥ 5-fold) in each of the 2 mutants compared to the wild-type strain. The largest group of genes downregulated in mhy1Δ mutant encodes cell wall proteins or enzymes involved in cell wall organization, suggesting that Mhy1 may regulate dimorphic transition by controlling these cell wall genes. We confirmed that the genes YALI0C23452, YALI0C15268 and YALI0B09955 are directly regulated by Mhy1. We also characterized the Mhy1 consensus binding site as 5'-WNAGGGG-3' (W = A or T; N = A, T, G or C). These results provide new insight in the functions of Msn2/Msn4-family transcription factors in fungi and the mechanism by which Mhy1 regulates dimorphic transition.

Enhanced mating-type switching and sexual hybridization in heterothallic yeast Yarrowia lipolytica.

Yarrowia lipolytica is a non-conventional, heterothallic, oleaginous yeast with wide range of industrial applications. Increasing ploidy can improve advantageous traits for industrial applications including genetic stability, stress resistance, and productivity, but the construction of knockout mutant strains from polyploid cells requires significant effort due to the increased copy numbers of target genes. The goal of this study was to evaluate the effectiveness of a mating-type switching strategy by single-step transformation without a genetic manipulation vestige, and to optimize the conventional method for increasing ploidy (mating) in Y. lipolytica. In this study, mating-type genes in haploid Y. lipolytica cells were scarlessly converted into the opposite type genes by site-specific homologous recombination, and the resulting MATB-type cells were mated at low temperature (22°C) with addition of sodium citrate with each MATA-type haploid cell to yield a MATA/MATB-type diploid strain with genetic information from both parental strains. The results of this study can be used to increase ploidy and for whole genome engineering of a yeast strain with unparalleled versatility for industrial application.

In Vitro Studies on Therapeutic Potential of Probiotic Yeasts Isolated from Various Sources.

The present study investigates the therapeutic properties of probiotic yeasts viz. Yarrowia lipolytica VIT-MN01, Kluyveromyces lactis VIT-MN02, Lipomyces starkeyi VIT-MN03, Saccharomycopsis fibuligera VIT-MN04 and Brettanomyces custersianus VIT-MN05. The antimutagenic activity of probiotic yeasts against the mutagens viz. Benzo[a]pyrene (B[a]P), and Sodium azide (SA) was tested. S. fibuligera VIT-MN04 showed highest antimutagenicity (75%). Binding ability on the mutagen acridine orange (AO) was tested and L. starkeyi VIT-MN03 was able to bind AO effectively (88%). The probiotic yeasts were treated with the genotoxins viz. 4-Nitroquinoline 1-Oxide (NQO) and Methylnitronitrosoguanidine (MNNG). The prominent changes in UV shift confirmed the reduction in genotoxic activity of S. fibuligera VIT-MN04 and L. starkeyi VIT-MN03, respectively. Significant viability of probiotic yeasts was noted after being exposed to mutagens and genotoxins. The adhesion capacity and anticancer activity were also assessed using Caco-2 and IEC-6 cell lines. Adhesion ability was found to be more in IEC-6 cells and remarkable antiproliferative activity was noted in Caco-2 cells compared to normal cells. Further, antagonistic activity of probiotic yeasts was investigated against S. typhimurium which was found to be more in S. fibuligera VIT-MN04 and L. starkeyi VIT-MN03. The inhibition of α-glucosidase and α-amylase activity confirmed the antidiabetic activity of probiotic yeasts. Antioxidant activity was also tested using standard assays. Therefore, based on the results, it can be concluded that probiotic yeasts can serve as potential therapeutic agents for the prevention and treatment of colon cancer, type 2 diabetes and gastrointestinal infections.

Effects of a high-cultivation temperature on the physiology of three different Yarrowia lipolytica strains.

Despite the increasing relevance, ranging from academic research to industrial applications, only a limited number of non-conventional, oleaginous Yarrowia lipolytica strains are characterized in detail. Therefore, we analyzed three strains in regard to their metabolic and physiological properties, especially with respect to important characteristics of a production strain. By investigating different cultivation conditions and media compositions, similarities and differences between the distinct strain backgrounds could be derived. Especially sugar alcohol production, as well as an agglomeration of cells were found to be connected with growth at high temperatures. In addition, sugar alcohol production was independent of high substrate concentrations under these conditions. To investigate the genotypic basis of particular traits, including growth characteristics and metabolite concentrations, genomic analysis were performed. We found sequence variations for one third of the annotated proteins but no obvious link to all phenotypic features.

Heavy metal tolerance in marine strains of Yarrowia lipolytica.

Heavy metal tolerance of two marine strains of Yarrowia lipolytica was tested on solid yeast extract peptone dextrose agar plates. Based on minimum inhibitory concentration esteems, it is inferred that the two strains of Y. lipolytica were tolerant to heavy metals such as Pb(II), Cr(III), Zn(II), Cu(II), As(V), and Ni(II) ions. The impact of various heavy metal concentrations on the growth kinetics of Y. lipolytica was likewise assessed. With increased heavy metal concentration, the specific growth rate was reduced with delayed doubling time. Furthermore, biofilm development of both yeasts on the glass surfaces and in microtitre plates was assessed in presence of different heavy metals. In microtitre plates, a short lag phase of biofilm formation was noticed without the addition of heavy metals in yeast nitrogen base liquid media. A lag phase was extended over increasing metal concentrations of media. Heavy metals like Cr(VI), Cd(II), and As(V) are contrastingly influenced on biofilms' formation of microtitre plates. Other heavy metals did not much influence on biofilms development. Thus, biofilm formation is a strategy of Y. lipolytica under stress of heavy metals has significance in bioremediation process for recovery of heavy metals from contaminated environment.

A mitochondrial manganese superoxide dismutase involved in innate immunity is essential for the survival of Chlamys farreri.

Superoxide dismutase (SOD) ubiquitously found in both prokaryotes and eukaryotes functions as the first and essential enzyme in the antioxidant system. In the present study, a manganese SOD (designated as CfmtMnSOD) was cloned from Zhikong scallop Chlamys farreri. The complete cDNA sequence of CfmtMnSOD contained a 681 bp open reading frame (ORF), encoding a peptide of 226 amino acids. A SOD_Fe_N domain and a SOD_Fe_C domain were found in the deduced amino acid sequence of CfmtMnSOD. The mRNA transcripts of CfmtMnSOD were constitutively expressed in all the tested tissues, including gill, gonad, hepatopancreas, hemocytes, mantle and muscle, with the highest expression level in hemocytes. After the stimulation of Vibrio splendidus, Staphylococcus aureus and Yarrowia lipolytica, the mRNA transcripts of CfmtMnSOD in hemocytes all significantly increased. The purified rCfmtMnSOD protein exhibited Mn2+ dependent specific and low stable enzymatic activities. After Vibrio challenge, the cumulative mortality of CfmtMnSOD-suppressed scallops was significantly higher than those of control groups and the semi-lethal time for CfmtMnSOD-suppressed scallops was rather shorter than those of control groups either. Moreover, the final mortality rate of CfmtMnSOD-suppressed group was significant higher than those of control groups, even without Vibrio challenge. All these results indicated that CfmtMnSOD was efficient antioxidant enzyme involved in the innate immunity, and also essential for the survival of C. farreri.

Advances in synthetic biology of oleaginous yeast Yarrowia lipolytica for producing non-native chemicals.

Oleaginous yeast Yarrowia lipolytica is an important industrial host for the production of enzymes, oils, fragrances, surfactants, cosmetics, and pharmaceuticals. More recently, improved synthetic biology tools have allowed more extensive engineering of this yeast species, which lead to the production of non-native metabolites. In this review, we summarize the recent advances of genome editing tools for Y. lipolytica, including the application of CRISPR/Cas9 system and discuss case studies, where Y. lipolytica was engineered to produce various non-native chemicals: short-chain fatty alcohols and alkanes as biofuels, polyunsaturated fatty acids for nutritional and pharmaceutical applications, polyhydroxyalkanoates and dicarboxylic acids as precursors for biodegradable plastics, carotenoid-type pigments for food and feed, and campesterol as a precursor for steroid drugs.

Impacts of environmental conditions on product formation and morphology of Yarrowia lipolytica.

The yeast Yarrowia lipolytica is an industrially important microorganism with distinctive physiological and metabolic characteristics. A variety of external factors (e.g., pH, temperature, and nutrient availability) influences the behavior of the yeast and may act as stress conditions which the cells must withstand and adapt. In this mini review, the impacts of environmental factors on the morphology and metabolite production by Y. lipolytica are summarized. In this regard, detailed insights into the effectors involved in the dimorphic transition of Y. lipolytica, the cultivation conditions employed, as well as the methods applied for the morphological characterization are highlighted. Concerning the metabolism products, a special focus is addressed on lipid and citric acid metabolites which have attracted significant attention in recent years. The dependence of lipid and citric acid productivity on key process parameters, such as media composition and physico-chemical variables, is thoroughly discussed. This review attempts to provide a recent update on the topic and will serve as a meaningful resource for researchers working in the field.

Investigating Proteome and Transcriptome Defense Response of Apples Induced by Yarrowia lipolytica.

A better understanding of the mode of action of postharvest biocontrol agents on fruit surfaces is critical for the advancement of successful implementation of postharvest biocontrol products. This is due to the increasing importance of biological control of postharvest diseases over chemical and other control methods. However, most of the mechanisms involved in biological control remain unknown and need to be explored. Yarrowia lipolytica significantly inhibited blue mold decay of apples caused by Penicillium expansum. The findings also demonstrated that Y. lipolytica stimulated the activities of polyphenoloxidase, peroxidase, chitinase, l-phenylalanine ammonia lyase involved in enhancing defense responses in apple fruit tissue. Proteomic and transcriptomic analysis revealed a total of 35 proteins identified as up- and down-regulated in response to the Y. lipolytica inducement. These proteins were related to defense, biotic stimulus, and stress responses, such as pathogenesis-related proteins and dehydrin. The analysis of the transcriptome results proved that the induced resistance was mediated by a crosstalk between salicylic acid (SA) and ethylene/jasmonate (ET/JA) pathways. Y. lipolytica treatment activated the expression of isochorismate synthase gene in the SA pathway, which up-regulates the expression of PR4 in apple. The expression of 1-aminocyclopropane-1-carboxylate oxidase gene and ET-responsive transcription factors 2 and 4, which are involved in the ET pathway, were also activated. In addition, cytochrome oxidase I, which plays an important role in JA signaling for resistance acquisition, was also activated. However, not all of the genes had a positive effect on the SA and ET/JA signal pathways. As transcriptional repressors in JA signaling, TIFY3B and TIFY11B were triggered by the yeast, but the gene expression levels were relatively low. Taken together, Y. lipolytica induced the SA and ET/JA signal mediating the defense pathways by stimulating defense response genes, such as peroxidase, thaumatin-like protein, and chitinase 4-like, which are involved in defense response in apple. [Formula: see text]

Engineering oxidative stress defense pathways to build a robust lipid production platform in Yarrowia lipolytica.

Microbially derived lipids have recently attracted renewed interests due to their broad applications in production of green diesels, cosmetic additives, and oleochemicals. Metabolic engineering efforts have targeted a large portfolio of biosynthetic pathways to efficiently convert sugar to lipids in oleaginous yeast. In the engineered overproducing strains, endogenous cell metabolism typically generates harmful electrophilic molecules that compromise cell fitness and productivity. Lipids, particularly unsaturated fatty acids, are highly susceptible to oxygen radical attack and the resulting oxidative species are detrimental to cell metabolism and limit lipid productivity. In this study, we investigated cellular oxidative stress defense pathways in Yarrowia lipolytica to further improve the lipid titer, yield, and productivity. Specifically, we determined that coupling glutathione disulfide reductase and glucose-6-phosphate dehydrogenase along with aldehyde dehydrogenase are efficient solutions to combat reactive oxygen and aldehyde stress in Y. lipolytica. With the reported engineering strategies, we were able to synchronize cell growth and lipid production, improve cell fitness and morphology, and achieved industrially-relevant level of lipid titer (72.7 g/L), oil content (81.4%) and productivity (0.97 g/L/h) in controlled bench-top bioreactors. The strategies reported here represent viable steps in the development of sustainable biorefinery platforms that potentially upgrade low value carbons to high value oleochemicals and biofuels. Biotechnol. Bioeng. 2017;114: 1521-1530. © 2017 Wiley Periodicals, Inc.

Analysis of virulence factors and in vivo biofilm-forming capacity of Yarrowia lipolytica isolated from patients with fungemia.

Yarrowia lipolytica is ubiquitous in the environment, opportunistic, and might be considered as one of the causative agents of catheter-related candidemia. Our work aimed to study some virulence factors of Y. lipolytica such as hydrolases production and biofilm formation with comparison to the most frequent Candida specie in human disease. In sum, 58 clinical isolates of Y. lipolytica, 16 C. glabrata, and 12 C. albicans were collected from Intensive care unit (ICU). All were tested for enzymatic production and biofilm formation. All tested isolates of C. albicans and C. glabrata were able to degrade casein, and 98.2% of Y. lipolytica showed caseinase activity but no gelatinase activity was detected in all isolates. Y. lipolytica strains showed significantly lower (3.4%) in vitro phospholipase activity than C. albicans and C. glabrata (P < .05). No significant differences of the hemolytic activity were detected between the three species (P > .05). Concerning biofilm formation, and unlike the results obtained on polystyrene plate, the number of adhered and biofilm cultivable cells obtained by Y. lipolytica after 168 hours of catheter subcutaneous implantation is significantly greater and tends to be more compact and structured hyphal layer. Although C. albicans remains the most pathogenic yeast, development of selective ability of Y. lipolytica to adhere, to form a biofilm on catheter medical devices, and to produce phospholipase and hemolytic enzyme is of particular interest, and it is strongly recommended to be vigilant in the use of medical implanted medical devices, particularly in ICU.

Dynamic behavior of Yarrowia lipolytica in response to pH perturbations: dependence of the stress response on the culture mode.

Yarrowia lipolytica, a non-conventional yeast with a promising biotechnological potential, is able to undergo metabolic and morphological changes in response to environmental conditions. The effect of pH perturbations of different types (pulses, Heaviside) on the dynamic behavior of Y. lipolytica W29 strain was characterized under two modes of culture: batch and continuous. In batch cultures, different pH (4.5, 5.6 (optimal condition), and 7) were investigated in order to identify the pH inducing a stress response (metabolic and/or morphologic) in Y. lipolytica. Macroscopic behavior (kinetic parameters, yields, viability) of the yeast was slightly affected by pH. However, contrary to the culture at pH 5.6, a filamentous growth was induced in batch experiments at pH 4.5 and 7. Proportions of the filamentous subpopulation reached 84 and 93 % (v/v) under acidic and neutral conditions, respectively. Given the significant impact of neutral pH on morphology, pH perturbations from 5.6 to 7 were subsequently assayed in batch and continuous bioreactors. For both process modes, the growth dynamics remained fundamentally unaltered during exposure to stress. Nevertheless, morphological behavior of the yeast was dependent on the culture mode. Specifically, in batch bioreactors where cells proliferated at their maximum growth rate, mycelia were mainly formed. Whereas, in continuous cultures at controlled growth rates (from 0.03 to 0.20 h-1) even closed to the maximum growth rate of the stain (0.24 h-1), yeast-like forms predominated. This pointed out differences in the kinetic behavior of filamentous and yeast subpopulations, cell age distribution, and pH adaptive mechanisms between both modes of culture.

Combining metabolic engineering and process optimization to improve production and secretion of fatty acids.

Microbial oils are sustainable alternatives to petroleum for the production of chemicals and fuels. Oleaginous yeasts are promising source of oils and Yarrowia lipolytica is the most studied and engineered one. Nonetheless the commercial production of biolipids is so far limited to high value products due to the elevated production and extraction costs. In order to contribute to overcoming these limitations we exploited the possibility of secreting lipids to the culture broth, uncoupling production and biomass formation and facilitating the extraction. We therefore considered two synthetic approaches, Strategy I where fatty acids are produced by enhancing the flux through neutral lipid formation, as typically occurs in eukaryotic systems and Strategy II where the bacterial system to produce free fatty acids is mimicked. The engineered strains, in a coupled fermentation and extraction process using alkanes, secreted the highest titer of lipids described so far, with a content of 120% of DCW.

Production of 1-decanol by metabolically 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 (<10mg/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 500mg/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.

Metabolic engineering of Yarrowia lipolytica to produce chemicals and fuels from xylose.

Yarrowia lipolytica is a biotechnological chassis for the production of a range of products, such as microbial oils and organic acids. However, it is unable to consume xylose, the major pentose in lignocellulosic hydrolysates, which are considered a preferred carbon source for bioprocesses due to their low cost, wide abundance and high sugar content. Here, we engineered Y. lipolytica to metabolize xylose to produce lipids or citric acid. The overexpression of xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis were necessary but not sufficient to permit growth. The additional overexpression of the endogenous xylulokinase enabled identical growth as the wild type strain in glucose. This mutant was able to produce up to 80g/L of citric acid from xylose. Transferring these modifications to a lipid-overproducing strain boosted the production of lipids from xylose. This is the first step towards a consolidated bioprocess to produce chemicals and fuels from lignocellulosic materials.

Metabolic evolution and (13) C flux analysis of a succinate dehydrogenase deficient strain of Yarrowia lipolytica.

Bio-based succinic acid production can redirect industrial chemistry processes from using limited hydrocarbons to renewable carbohydrates. A fermentation process that does not require pH-titrating agents will be advantageous to the industry. Previously, a Yarrowia lipolytica strain that was defective for succinate dehydrogenase was constructed and was found to accumulate up to 17.5 g L(-1) of succinic acid when grown on glycerol without buffering. Here, a derivative mutant was isolated that produced 40.5 g L(-1) of succinic acid in 36 h with a yield of 0.32 g g(-1) glycerol. A combination approach of induced mutagenesis and metabolic evolution allowed isolation of another derivative that could utilize glucose efficiently and accumulated 50.2 g L(-1) succinic acid in 54 h with a yield of 0.43 g g(-1) . The parent strain of these isolated mutants was used for [1,6-(13) C2 ]glucose assimilation analysis. At least 35% glucose was estimated to be utilized through the pentose phosphate pathway, while ≥84% succinic acid was formed through the oxidative branch of the tricarboxylic acid cycle. Biotechnol. Bioeng. 2016;113: 2425-2432. © 2016 Wiley Periodicals, Inc.

The effect of yeast Yarrowia lipolytica on the antioxidant indices and macro-and microelements in blood plasma of turkey hens.

The aim of the study was to assess the effect of different amounts of Yarrowia lipolytica yeast on the redox response and content of macro- and microelements in the blood plasma of turkey hens. The experiment was carried out on 240 turkey hens aged from 1 to 16 weeks. The birds were randomly assigned to 3 experimental groups of 80 birds each. Group I served as a control (K) and did not receive any experimental compounds. The turkey hens from the experimental groups (YL3 and YL6) were administered dried Yarrowia lipolytica yeast in their feed mixtures in the amount of 3% (YL3) or 6% (YL6). Yarrowia lipolytica yeast in the feed mixtures for the turkey hens did not induce oxidation reactions in the organism of the birds. However, an increase in catalase activity and a reduction in the level of LOOH, MDA and vitamin C were observed in the blood plasma of the turkey hens whose diet was supplemented with YL yeast. In the case of other indices, such as superoxide dismutase activity and total antioxidant potential (FRAP), the additive caused no significant changes. Administering Yarrowia lipolytica yeast to turkey hens may stimulate the enzymatic response of the antioxidant system (e.g. increasing catalase activity), mainly by increasing the concentration of iron in the plasma.

[Antistress systems of the yeast Yarrowia lipolitica (review)].

The authors' and literature data on the adaptation response of the micromycetes Yarrowia lipolytica to various stress impacts are considered in the review. The uniformity of cellular response to all stress factors is discussed.