Synthesis of novel benzylamine antimycotics and evaluation of their antimycotic potency.

A series of 23 novel benzylamines was synthesized by reductive amination from halogen-substituted 3- and 4-benzyloxybenzaldehyde derivatives and 6-methylhept-2-yl amine or n-octylamine. The antimycotic activity of the resulting amines was evaluated in a microdilution assay against the apathogenic yeast Yarrowia lipolytica as test microorganism. Promising compounds were also tested against human pathogenic Candida species. The influence of halogen substituents at the benzyl ether side chain was studied in this screening, as well as the influence of the branched side chain of (±)-6-methylhept-2-yl amine in comparison with the n-octyl side chain.

Targeted Integration Through Transformation of Hydroxyurea-Arrested Cells.

Homologous recombination is required to specifically target DNA to a desired genomic locus. Non-homologous end joining is the predominant form of recombination in Yarrowia lipolytica. Transformation of this organism with linear DNA therefore results in random integration of the introduced DNA into the genome. In this protocol, hydroxyurea-mediated cell cycle arrest is applied to significantly increase the rate of homologous recombination during transformation and enhance targeted integration.

Tolerance of Yarrowia lipolytica to inhibitors commonly found in lignocellulosic hydrolysates.

BACKGROUND: Lignocellulosic material is a suitable renewable carbon and energy source for microbial cell factories, such as Yarrowia lipolytica. To be accessible for microorganisms, the constituent sugars need to be released in a hydrolysis step, which as a side effect leads to the formation of various inhibitory compounds. However, the effects of these inhibitory compounds on the growth of Y. lipolytica have not been thoroughly investigated. RESULTS: Here we show the individual and combined effect of six inhibitors from three major inhibitor groups on the growth of Y. lipolytica. We engineered a xylose consuming strain by overexpressing the three native genes XR, XDH, and XK and found that the inhibitor tolerance of Y. lipolytica is similar in glucose and in xylose. Aromatic compounds could be tolerated at high concentrations, while furfural linearly increased the lag phase of the cultivation, and hydroxymethylfurfural only inhibited growth partially. The furfural induced increase in lag phase can be overcome by an increased volume of inoculum. Formic acid only affected growth at concentrations above 25 mM. In a synthetic hydrolysate, formic acid, furfural, and coniferyl aldehyde were identified as the major growth inhibitors. CONCLUSION: We showed the individual and combined effect of inhibitors found in hydrolysate on the growth of Y. lipolytica. Our study improves understanding of the growth limiting inhibitors found in hydrolysate and enables a more targeted engineering approach to increase the inhibitor tolerance of Y. lipolytica. This will help to improve the usage of Y. lipolytica as a sustainable microbial cell factory.

HOG-Independent Osmoprotection by Erythritol in Yeast Yarrowia lipolytica.

Erythritol is a polyol produced by Yarrowia lipolytica under hyperosmotic stress. In this study, the osmo-sensitive strain Y. lipolytica yl-hog1Δ was subjected to stress, triggered by a high concentration of carbon sources. The strain thrived on 0.75 M erythritol medium, while the same concentrations of glucose and glycerol proved to be lethal. The addition of 0.1 M erythritol to the medium containing 0.75 M glucose or glycerol allowed the growth of yl-hog1Δ. Supplementation with other potential osmolytes such as mannitol or L-proline did not have a similar effect. To examine whether the osmoprotective effect might be related to erythritol accumulation, we deleted two genes involved in erythritol utilization, the transcription factor Euf1 and the enzyme erythritol dehydrogenase Eyd1. The strain eyd1Δ yl hog1Δ, which lacked the erythritol utilization enzyme, reacted to the erythritol supplementation significantly better than yl-hog1Δ. On the other hand, the strain euf1Δ yl-hog1Δ became insensitive to supplementation, and the addition of erythritol could no longer improve the growth of this strain in hyperosmotic conditions. This indicates that Euf1 regulates additional, still unknown genes involved in erythritol metabolism.

Yarrowia lipolytica causes sporadic cases and local outbreaks of infections and colonisation.

BACKGROUND: Yarrowia lipolytica belongs to the normal human microbiota but is also found in substrates with high contents in lipids and used in biotechnological processes. It is sometimes reported as human pathogen and especially in catheter-related candidaemia. OBJECTIVES: Two apparently grouped cases of infections and/or contamination were reported involving 3 and 9 patients, respectively, in two hospitals. The goal of this study was to design a molecular tool to study the genetic diversity of Y lipolytica and confirm or not the common source of contamination during these grouped cases. METHODS: Given that there is no genotyping method, we used genomic markers assessed on environmental isolates to determine intra-species relationship. We selected five highly polymorphic intergenic regions, totalling more than 3200 bp and sequenced them for clinical (n = 20) and environmental (n = 14) isolates. Antifungal susceptibility was determined by EUCAST broth microdilution method. RESULTS: Multiple alignment of the five sequences revealed divergence of 0%-5.8% between isolates as compared to approximately 0.2%-0.25% after alignment of whole genomes, suggesting their potential usefulness to establish genetic relatedness. The analysis showed the multiple origins of the isolates. It uncovered two grouped case of fungaemia involving 3 and 2 patients, respectively. It also revealed several unrelated sporadic cases despite their temporal relationship and one probable laboratory contamination by a common yet uncovered source, explaining several consecutive positive cultures without infection. All isolates had high minimal inhibitory concentration (MIC) for flucytosine, the majority (14/34) was susceptible to fluconazole, and all to the other antifungal agents tested. CONCLUSION: This method could help elucidate cases related to the opportunistic pathogen Y lipolytica.

Pregnenolone Overproduction in Yarrowia lipolytica by Integrative Components Pairing of the Cytochrome P450scc System.

Microbial production of steroid drugs exhibits great potentials in much greener and more sustainable manners, in which engineering multiple cytochrome P450s is the prerequisite requirement. The pairing of multicomponents of P450 systems is a tremendous challenge. Herein, biosynthesis of pregnenolone (a common precursor of steroid drugs) in Yarrowia lipolytica was taken as a typical instance to explore the engineering strategy of the cytochrome P450 side-chain cleavage enzyme (P450scc) system. The mature forms of the components belonging to P450scc system, CYP11A1, adrenodoxin (Adx), and adrenodoxin reductase (AdR), were coexpressed in a former constructed campesterol producing strain. To maximize pregnenolone production, an integrative components pairing strategy was proposed for pairing the component sources and balancing the expression levels of CYP11A1, Adx, and AdR. Led by the above approaches, a 2344-fold improvement of pregnenolone titer was achieved at the shake flask level. Consequently, a highest reported pregnenolone titer of 78.0 mg/L in microbes was obtained in a 5 L bioreactor. Our study not only highlights the integrative components pairing of cytochrome P450scc as a general strategy for engineering other cytochrome P450s, but also provides a feasible and efficient platform of Y. lipolytica for other steroids production.

Investigating the Influence of Glycerol on the Utilization of Glucose in Yarrowia lipolytica Using RNA-Seq-Based Transcriptomics.

Glycerol is considered as a promising substrate for biotechnological applications and the non-conventional yeast Yarrowia lipolytica has been used extensively for the valorization of this compound. Contrary to S. cerevisiae, Y. lipolytica seems to prefer glycerol over glucose and it has been reported previously that the presence of glycerol can suppress the consumption of glucose in co-substrate fermentations. Based on these observations, we hypothesized glycerol repression-like effects in Y. lipolytica, which are converse to well described carbon repression mechanisms ensuring the prioritized use of glucose (e.g., in S. cerevisiae). We therefore aimed to investigate this effect on the level of transcription. Strains varying in the degree of glucose suppression were chosen and characterized in high-resolution growth screenings, resulting in the detection of different growth phenotypes under glycerol-glucose mixed conditions. Two strains, IBT and W29, were selected and cultivated in chemostats using glucose, glycerol and glucose/glycerol as carbon sources, followed by an RNA-Seq-based transcriptome analysis. We could show that several transporters were significantly higher expressed in W29, which is potentially related to the observed physiological differences. However, most of the expression variation between the strains were regardless of the carbon source applied, and cross-comparisons revealed that the strain-specific carbon source responses underwent in the opposite direction. A deeper analysis of the substrate specific carbon source response led to the identification of several differentially expressed genes with orthologous functions related to signal transduction and transcriptional regulation. This study provides an initial investigation on potentially novel carbon source regulation mechanisms in yeasts.

Genotoxic effect of caffeine in Yarrowia lipolytica cells deficient in DNA repair mechanisms.

Caffeine is a compound that can exert physiological-beneficial effects in the organism. Nevertheless, there are controversies about its protective-antioxidant and/or its negative genotoxic effect. To abound on the analysis of the possible genotoxic/antioxidant effect of caffeine, we used as research model the yeast Yarrowia lipolytica parental strain, and mutant strains (∆rad52 and ∆ku80), which are deficient in the DNA repair mechanisms. Caffeine (5 mM) showed a cytostatic effect on all strains, but after 72 h of incubation the parental and ∆ku80 strains were able to recover of this inhibitory effect on growth, whereas ∆rad52 was unable to recover. When cells were pre-incubated with caffeine and H2O2 or incubated with a mixture of both agents, a higher inhibitory effect on growth of mutant strains was observed and this effect was noticeably greater for the Δrad52 strain. The toxic effect of caffeine appears to be through a mechanism of DNA damage (genotoxic effect) that involves DSB generation since, in all tested conditions, the growth of Δrad52 strain (cells deficient in HR DNA repair mechanism) was more severely affected.

Development of antifungal ingredients for dairy products: From in vitro screening to pilot scale application.

Biopreservation represents a complementary approach to traditional hurdle technologies for reducing microbial contaminants (pathogens and spoilers) in food. In the dairy industry that is concerned by fungal spoilage, biopreservation can also be an alternative to preservatives currently used (e.g. natamycin, potassium sorbate). The aim of this study was to develop antifungal fermentates derived from two dairy substrates using a sequential approach including an in vitro screening followed by an in situ validation. The in vitro screening of the antifungal activity of fermentates derivating from 430 lactic acid bacteria (LAB) (23 species), 70 propionibacteria (4 species) and 198 fungi (87 species) was performed against four major spoilage fungi (Penicillium commune, Mucor racemosus, Galactomyces geotrichum and Yarrowia lipolytica) using a cheese-mimicking model. The most active fermentates were obtained from Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei/paracasei and Lactobacillus plantarum among the tested LAB, Propionibacterium jensenii among propionibacteria, and Mucor lanceolatus among the tested fungi. Then, for the 11 most active fermentates, culture conditions were optimized by varying incubation time and temperature in order to enhance their antifungal activity. Finally, the antifungal activity of 3 fermentates of interest obtained from Lactobacillus rhamnosus CIRM-BIA1952, P. jensenii CIRM-BIA1774 and M. lanceolatus UBOCC-A-109193 were evaluated in real dairy products (sour cream and semi-hard cheese) at a pilot-scale using challenge and durability tests. In parallel, the impact of these ingredients on organoleptic properties of the obtained products was also assessed. In semi-hard cheese, application of the selected fermentates on the cheese surface delayed the growth of spoilage molds for up to 21 days, without any effect on organoleptic properties, P. jensenii CIRM-BIA1774 fermentate being the most active. In sour cream, incorporation of the latter fermentate at 2 or 5% yielded a high antifungal activity but was detrimental to the product organoleptic properties. Determination of the concentration limit, compatible with product acceptability, showed that incorporation of this fermentate at 0.4% prevented growth of fungal contaminants in durability tests but had a more limited effect against M. racemosus and P. commune in challenge tests. To our knowledge, this is the first time that the workflow followed in this study, from in vitro screening using dairy matrix to scale-up in cheese and sour cream, is applied for production of natural ingredients relying on a large microbial diversity in terms of species and strains. This approach allowed obtaining several antifungal fermentates which are promising candidates for dairy products biopreservation.

Efficient Conversion of Cane Molasses Towards High-Purity Isomaltulose and Cellular Lipid Using an Engineered Yarrowia lipolytica Strain in Fed-Batch Fermentation.

: Cane molasses is one of the main by-products of sugar refineries, which is rich in sucrose. In this work, low-cost cane molasses was introduced as an alternative substrate for isomaltulose production. Using the engineered Yarrowia lipolytica, the isomaltulose production reached the highest (102.6 g L-¹) at flask level with pretreated cane molasses of 350 g L-¹ and corn steep liquor of 1.0 g L-¹. During fed-batch fermentation, the maximal isomaltulose concentration (161.2 g L-¹) was achieved with 0.96 g g-¹ yield within 80 h. Simultaneously, monosaccharides were completely depleted, harvesting the high isomaltulose purity (97.4%) and high lipid level (12.2 g L-¹). Additionally, the lipids comprised of 94.29% C16 and C18 fatty acids, were proved suitable for biodiesel production. Therefore, the bioprocess employed using cane molasses in this study was low-cost and eco-friendly for high-purity isomaltulose production, coupling with valuable lipids.

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.

Yarrowia lipolytica: a beneficious yeast in biotechnology as a rare opportunistic fungal pathogen: a minireview.

Yarrowia lipolytica is one of the most studied "non-conventional" yeast species capable of synthesizing a wide group of valuable metabolites, in particular lipases and other hydrolytic enzymes, microbial oil, citric acid, erythritol and γ-decalactone. Processes based on the yeast have GRAS status ("generally recognized as safe") given by Food and Drug Administration. The majority of research communications regarding to Y. lipolytica claim that the yeast species is non-pathogenic. In spite of that, Y. lipolytica, like other fungal species, can cause infections in immunocompromised and critically ill patients. The yeast possess features that facilitate invasion of the host cell (particularly production of hydrolytic enzymes), as well as the protection of the own cells, such as biofilm formation. The aim of this study was to present well-known yeast species Y. lipolytica as a rare opportunistic fungal pathogen. Possible pathogenicity and epidemiology of this yeast species were discussed. Antifungal drugs susceptibility and increasing resistance to azoles in Y. lipolytica yeasts were also presented.

Determination of the effect of polyamines on an oil-degrading strain of Yarrowia lipolytica using an odc minus mutant.

Yarrowia lipolytica is an ascomycetous dimorphic yeast with immense potential for industrial applications, including bioremediation of crude oil-contaminated environments. It has been shown that a dimorphic marine isolate of Y. lipolytica (var. indica) has significant capacity to degrade fatty acids and alkanes, when in its yeast morphology. It has also been demonstrated that polyamines play an important role in the yeast-to-mycelium transition of different strains of Y. lipolytica that are unable to utilize those carbon sources. To determine the role of polyamines on their capacity to utilize oils and hydrocarbons, on the dimorphic transition, and also on other characteristics of the var. indica strain of Y. lipolytica, we proceeded to obtain ornithine decarboxylase minus (odc-) mutants. These mutants behaved as yeasts independently of the concentrations of putrescine added. Further, they conserved the oil-degrading capacity of the parent strain. The odc- mutant can thus be used in fatty acid degradation, and oil spill remediation with distinct advantages.

Overproduction of Fatty Acid Ethyl Esters by the Oleaginous Yeast Yarrowia lipolytica through Metabolic Engineering and Process Optimization.

Recent advances in the production of biofuels by microbes have attracted attention due to increasingly limited fossil fuels. Biodiesels, especially fatty acid ethyl esters (FAEEs), are considered a potentially fully sustainable fuel in the near future due to similarities with petrodiesels and compatibility with existing infrastructure. However, biosynthesis of FAEEs is limited by the supply of precursor lipids and acetyl-CoA. In the present study, we explored the production potential of an engineered biosynthetic pathway coupled to the addition of ethanol in the oleaginous yeast Yarrowia lipolytica. This type of yeast is able to supply a greater amount of precursor lipids than species typically used. To construct the FAEEs synthesis pathway, WS genes that encode wax ester synthases (WSs) from different species were codon-optimized and heterologously expressed in Y. lipolytica. The most productive engineered strain was found to express a WS gene from Marinobacter hydrocarbonoclasticus strain DSM 8798. To stepwisely increase FAEEs production, we optimized the promoter of WS overexpression, eliminated ß-oxidation by deleting the PEX10 gene in our engineered strains, and redirected metabolic flux toward acetyl-CoA. The new engineered strain, coupled with an optimized ethanol concentration, led to an approximate 5.5-fold increase in extracellular FAEEs levels compared to the wild-type strain and a maximum FAEEs titer of 1.18 g/L in shake flask cultures. In summary, the present study demonstrated that an engineered Y. lipolytica strain possessed a high capacity for FAEEs production and may serve as a platform for more efficient biodiesel production in the future.

Morphological response of Yarrowia lipolytica under stress of heavy metals.

The marine dimorphic yeast Yarrowia lipolytica has been proposed as a suitable model for the dimorphism study. In this study, the morphological behaviour of two marine strains of Y. lipolytica (NCIM 3589 and NCIM 3590) was studied under stress of different heavy metals. Scanning electron microscopy was used to investigate the morphological features of yeast cells. This study revealed that the normal ellipsoidal shape of yeast cells was changed into oval, rounded, or elongated in response to different heavy-metal stress. Light microscopy was also used to investigate individual properties of yeast cells. The average cell length and radius of both marine strains was increased with increasing concentrations of heavy-metal ions. In addition, the elongation factor was calculated and was increased in the presence of heavy metals like Pb(II), Co(II), Cr(III), Cr(VI), and Zn(II) under the static conditions.

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 metabolic engineering strategy for producing free fatty acids by the Yarrowia lipolytica yeast based on impairment of glycerol metabolism.

In recent years, bio-based production of free fatty acids from renewable resources has attracted attention for their potential as precursors for the production of biofuels and biochemicals. In this study, the oleaginous yeast Yarrowia lipolytica was engineered to produce free fatty acids by eliminating glycerol metabolism. Free fatty acid production was monitored under lipogenic conditions with glycerol as a limiting factor. Firstly, the strain W29 (Δgpd1), which is deficient in glycerol synthesis, was obtained. However, W29 (Δgpd1) showed decreased biomass accumulation and glucose consumption in lipogenic medium containing a limiting supply of glycerol. Analysis of substrate utilization from a mixture of glucose and glycerol by the parental strain W29 revealed that glycerol was metabolized first and glucose utilization was suppressed. Thus, the Δgpd1Δgut2 double mutant, which is deficient also in glycerol catabolism, was constructed. In this genetic background, growth was repressed by glycerol. Oleate toxicity was observed in the Δgpd1Δgut2Δpex10 triple mutant strain which is deficient additionally in peroxisome biogenesis. Consequently, two consecutive rounds of selection of spontaneous mutants were performed. A mutant released from growth repression by glycerol was able to produce 136.8 mg L-1 of free fatty acids in a test tube, whereas the wild type accumulated only 30.2 mg L-1 . Next, an isolated oleate-resistant strain produced 382.8 mg L-1 of free fatty acids. Finely, acyl-CoA carboxylase gene (ACC1) over-expression resulted to production of 1436.7 mg L-1 of free fatty acids. The addition of dodecane promoted free fatty acid secretion and enhanced the level of free fatty acids up to 2033.8 mg L-1 during test tube cultivation.

Improvement of extracellular lipase production by a newly isolated Yarrowia lipolytica mutant and its application in the biosynthesis of L-ascorbyl palmitate.

Yarrowia lipolytica Wt-11 producing an extracellular lipase was isolated and identified. To improve the lipase production, Y. lipolytica Wt-11 was subjected to low-energy ion implantation mutation breeding, and a best mutant, Y. lipolytica Mut-96, was obtained after screening. Under the optimal cultivation conditions, the scaled-up production of lipases were performed, and the lipase activity of Y. lipolytica Mut-96 was enhanced nearly 5.5-fold compared with that of Y. lipolytica Wt-11. After fermentation, the lipases were purified, and the characteristics of the purified lipases were studied. The optimum temperatures and pHs for lipases from Wt-11and Mut-96 were 30°C and 8.0, respectively. The purified lipases were stable between pH 7.0 and 8.5 and unstable at temperatures above 40°C. The lipase activities were enhanced by Ca2+, Ba2+, Mn2+, Fe2+ and SDS. The synthesis of L-ascorbyl palmitate via esterification with L-ascorbic acid and palmitic acid by immobilized lipases from Wt-11 and Mut-96 in organic media was investigated, and the L-ascorbyl palmitate can be respectively produced at levels of 14.8 and 27.5g/L.

Mutants of Yarrowia lipolytica NCIM 3589 grown on waste cooking oil as a biofactory for biodiesel production.

BACKGROUND: Oleaginous yeasts are fast emerging as a possible feedstock for biodiesel production. Yarrowia lipolytica, a model oleaginous yeast is known to utilize a variety of hydrophobic substrates for lipid accumulation including waste cooking oil (WCO). Approaches to increase lipid content in this yeast include metabolic engineering which requires manipulation of multiple genes in the lipid biosynthesis pathway. A classical and cost-effective approach, namely, random chemical mutagenesis on the yeast can lead to increased production of biodiesel as is explored here. RESULTS: In this study, chemical mutagenesis using the alkylating agent, N- methyl-N'-nitro-N-nitrosoguanidine (MNNG) as well as an additional treatment with cerulenin, a fatty acid synthase inhibitor generated 800 mutants of Y. lipolytica NCIM 3589 (761 MNNG treated and 39 MNNG + cerulenin treated). A three-stage screening using Sudan Black B plate technique, Nile red fluorimetry and total lipid extraction using solvent was performed, which enabled selection of ten high lipid yielding mutants. Time course studies of all the ten mutants were further undertaken in terms of biomass, lipid yield and lipid content to select three stable mutants (YlB6, YlC7 and YlE1) capable of growing and accumulating lipid on WCO, with lipid contents of 55, 60 and 67% as compared to 45% for the wild type. The mutants demonstrated increased volumetric lipid productivities (0.062, 0.044 and 0.041 g L-1 h-1) as compared to the wild type (0.033 g L-1 h-1). The fatty acid profile of the three mutants consisted of a high content of C16 and C18 saturated and monounsaturated fatty acids and was found to be suitable for biodiesel production. The fuel properties, namely, density, kinematic viscosity, total acid number, iodine value of the three mutants were evaluated and found to lie within the limits specified by internationally accepted standards. Additionally, it was noted that the mutants demonstrated better cetane numbers and higher heating values than the wild type strain. CONCLUSION: The chemical mutagenesis strategy adopted in this study resulted in the successful isolation of three stable high SCO yielding mutants. The mutants, namely, YlB6, YlC7 and YlE1 exhibited a 1.22, 1.33 and 1.49-fold increase in lipid contents when grown on 100 g L-1 waste cooking oil than the parental yeast strain. The fatty acid methyl ester (FAME) profiles of all the three mutants was determined to be suitable for biodiesel suggesting their potential applicability while simultaneously addressing the management of waste cooking oil.

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.