Yarrowia Lipolytica Cells Research Articles

Digital holographic microscopy is suitable for lipid accumulation analysis in single cells of Yarrowia lipolytica

Digital holographic microscopy is suitable for lipid accumulation analysis in single cells of Yarrowia lipolytica

Experimental validation off-line a nonlinear controller for removal of chromium using non-living cells of Yarrowia lipolytica

The removal of hexavalent chromium [Cr (VI)] using non-living cells of Yarrowia lipolytica was investigated. Batch and continuous studies on removal of Cr (VI) achieved 97% and 99% removal from aqueous phase, respectively. The specific uptake values at pH of 2 in batch process were 40.73 ± 1.3 mg/g and 30.09 ± 0.23 mg/g on non-living cells, when 100 and 200 mg/L of metal Cr (VI) concentrations were used. In order to investigate the regulation of Cr (VI) under continuous operation based on reaction volume numerically a new class of feedback controller from structure polynomial was designed. The proposed methodology was used to an experimentally kinetic model for a removal Cr (VI) from Yarrowia lipolytica biomass was showed satisfactory closed-loop performance the proposed controller. Starting from an off-line optimization performed in simulation, we present the controller implementation, focussing on the methodology required to could be suitable for implementation in real time. In our experimental results, we highlight some discrepancies between simulation and reality despite these differences, the controller managed to perform convergence to removal Cr (VI). Finally, the results validated with off-line samples suggest that the proposed control could be suitable for in application in potential scenarios for wastewater treatment.

Building Yarrowia lipolytica Cell Factories for Advanced Biomanufacturing: Challenges and Solutions.

Microbial cell factories have shown great potential for industrial production with the benefit of being environmentally friendly and sustainable. Yarrowia lipolytica is a promising and superior non-model host for biomanufacturing due to its cumulated advantages compared to model microorganisms, such as high fluxes of metabolic precursors (acetyl-CoA and malonyl-CoA) and its naturally hydrophobic microenvironment. However, although diverse compounds have been synthesized in Y. lipolytica cell factories, most of the relevant studies have not reached the level of industrialization and commercialization due to a number of remaining challenges, including unbalanced metabolic flux, conflict between cell growth and product synthesis, and cytotoxic effects. Here, various metabolic engineering strategies for solving the challenges are summarized, which is developing fast and extremely conducive to rational design and reconstruction of robust Y. lipolytica cell factories for advanced biomanufacturing. Finally, future engineering efforts for enhancing the production efficiency of this platform strain are highlighted.

Unconventional Extraction Methods of Oleaginous Yeast Cell Pretreatment and Disruption

Extraction is one of the most commonly used methods for obtaining and purifying chemical compounds for commercial usage. The aim of this study was to evaluate the effect of unconventional permeabilization and cell disruption methods on the yield of lipid extraction from cells of the oleaginous yeast Yarrowia lipolytica. Batch cultures in a medium with molasses and waste post-frying oil were carried out. The biomass was subjected to pulsed electric field (PEF), high-pressure processing (HPP), ultrasounds (US), and several conventional processing techniques with chemical and mechanical agents (glass beads, acetone, Triton and Tween surfactants). The effectiveness of the applied methods, either on cell permeabilization or cell disruption, was investigated by analyzing the oil and total protein extraction yield and oil leaching efficiency, as well as by using microscope images. The PEF and US treatments proved to be effective permeabilization methods as a step of sample pretreatment for extraction. These unconventional physical methods could efficiently increase intracellular lipid extraction yield in solvent applications.

Combination of microbial and chemical synthesis for the sustainable production of β-elemene, a promising plant-extracted anticancer compound.

Beta-elemene, a class of sesquiterpene derived from the Chinese medicinal herb Curcuma wenyujin, is widely used in clinical medicine due to its broad-spectrum antitumor activity. However, the unsustainable plant extraction prompted the search for environmentally friendly strategies for β-elemene production. In this study, we designed a Yarrowia lipolytica cell factory that can continuously produce germacrene A, which is further converted into β-elemene with 100% yield through a Cope rearrangement reaction by shifting the temperature to 250°C. First, the productivity of four plant-derived germacrene A synthases was evaluated. After that, the metabolic flux of the precursor to germacrene A was maximized by optimizing the endogenous mevalonate pathway, inhibiting the competing squalene pathway, and expressing germacrene A synthase gene in multiple copies. Finally, the most promising strain achieved the highest β-elemene titer reported to date with 5.08 g/L. This sustainable and green method has the potential for industrial β-elemene production.

Metabolome and Transcriptome Profiling Reveal Carbon Metabolic Flux Changes in Yarrowia lipolytica Cells to Rapamycin.

Yarrowia lipolytica is an oleaginous yeast for the production of oleochemicals and biofuels. Nitrogen deficiency is beneficial to lipids biosynthesis in Y. lipolytica. Target of rapamycin (TOR) regulates the utilization of nutrients, which is inhibited in nitrogen starvation or by rapamycin treatment. However, under nitrogen-rich conditions, the lipids biosynthesis in Y. lipolytica after inhibition of TOR by rapamycin is elusive. Combining metabolomics and transcriptomics analysis, we found that rapamycin altered multiple metabolic processes of Y. lipolytica grown in nitrogen-rich medium, especially the metabolisms of amino acids and lipids. A total of 176 differentially accumulated metabolites were identified after rapamycin treatment. Rapamycin increased the levels of tryptophan, isoleucine, proline, serine, glutamine, histidine, lysine, arginine and glutamic acid, and decreased the levels of threonine, tyrosine and aspartic acid. Two fatty acids in lipid droplets, stearic acid (down-regulated) and stearidonic acid (up-regulated), were identified. The expression of 2224 genes changed significantly after rapamycin treatment. Further analysis revealed that rapamycin reduced carbon flux through lipids biosynthesis, accompanied by increased carbon flux through fatty acids degradation and amino acid (especially glutamic acid, glutamine, proline and arginine) biosynthesis. The dataset provided here is valuable for understanding the molecular mechanisms of amino acid and lipids metabolisms in oleaginous yeast.

Biosynthesis of insect sex pheromone precursors via engineered β-oxidation in yeast.

Mating disruption with insect sex pheromones is an attractive and environmentally friendly technique for pest management. Several Lepidoptera sex pheromones have been produced in yeast, where biosynthesis could be accomplished by the expression of fatty acyl-CoA desaturases and fatty acyl-CoA reductases. In this study, we aimed to develop yeast Yarrowia lipolytica cell factories for producing Lepidoptera pheromones which biosynthesis additionally requires β-oxidation, such as (Z)-7-dodecenol (Z7-12:OH), (Z)-9-dodecenol (Z9-12:OH), and (Z)-7-tetradecenol (Z7-14:OH). We expressed fatty acyl-CoA desaturases from Drosophila melanogaster (Dmd9) or Lobesia botrana (Lbo_PPTQ) and fatty acyl-CoA reductase from Helicoverpa armigera (HarFAR) in combinations with 11 peroxisomal oxidases of different origins. Yeast cultivations were performed with supplementation of methyl myristate (14:Me). The oxidase Lbo_31670 from L. botrana provided the highest titers of (Z)-7-dodecenoate, (Z)-9-dodecenoate, and (Z)-7-tetradecenoate. However, no chain-shortened fatty alcohols were produced. The mutation of fatty acid synthase (Fas2pI1220F) to increase myristate production did not lead to targeted fatty alcohol production. The problem was solved by directing the reductase into peroxisomes, where the strain with Dmd9 produced 0.10±0.02mg/lof Z7-12:OH and 0.48±0.03mg/lof Z7-14:OH, while the strain with Lbo_PPTQ produced 0.21±0.03mg/lof Z9-12:OH and 0.40±0.07mg/lof Z7-14:OH. In summary, the engineering of β-oxidation in Y. lipolytica allowed expanding the portfolio of microbially produced insect sex pheromones.

A Feed Additive Containing Encapsulated 6-Phytase within Recombinant Yarrowia lipolytica Cells Produced by Cultivation on Fat-Containing Waste

Feed phytases are purchased as a dry culture medium of secreting producers, mostly micellar fungi. These preparations are required to withstand heating up to 75–80 °C because they are intended for mixing with feed components with subsequent granulation by spray drying. For this reason, many phytases that have a high specific activity at 37 °C and correspond to the optimal pH of intestinal chyme are not used in practice. A novel expression system allowing accumulation of the phytase from Obesumbacterium proteus within yeast Yarrowia lipolytica was proposed. Encapsulation increases thermal stability of the enzyme from 55 °C up to 70 °C. The obtained preparation exhibited a high impact on the daily weight gain of a weaned mouse model fed a phosphorus-deficient diet at a dosage 165 phytase activity units (FYT)/kg, whereas a commercial phytase preparation—Ladozyme Proxi derived from Aspergillus ficuum—did not improve the daily weight gain even at the dosage of 15,000 FYT/kg.

Raman-probes for monitoring metabolites and nutrient fate in Yarrowia lipolytica using deuterated glucose

The aim of this study was to explore the application of deuterium (D) labeled glucose-D 7 as Raman-tag in Yarrowia lipolytica to assess triacyl glycerides (TAG) biosynthesis. Cells were grown in N depleted media in: (i) glucose in H 2 O (control), (ii) glucose-D 7 in H 2 O and (iii) glucose-D 7 in D 2 O, and the resulting biomass and lipids quantified. Infrared and Raman spectroscopies showed the incorporation of D with the presence of a C-D stretching bands in the silent region (1800–2600 cm −1 ) for cells grown in glucose-D 7 in H 2 O and glucose-D 7 in D 2 O as compared to the control. Fatty acid methyl ester analysis of cells grown in the three media showed significant differences in lipid profiles. Cells grown on glucose-D 7 in H 2 O and glucose-D 7 in D 2 O showed unequivocally D incorporation in fatty acids and TAG by mass spectrometry. Higher D incorporation was observed when cells were grown in D 2 O than H 2 O. Deuterium was also incorporated into the cell wall polysaccharides. • Deuterium was incorporated in Yarrowia lipolytica cells that were fed glucose-D 7 . • Deuterium was incorporated in both the lipids and cell wall polysaccharides. • Higher levels of D were observed when using D 2 O versus H 2 O. • The lipids and polysaccharides were analyzed by Raman, FTIR, GC-MS and ESI-MS.

Graphene coated magnetic nanoparticles facilitate the release of biofuels and oleochemicals from yeast cell factories

Engineering of microbial cells to produce high value chemicals is rapidly advancing. Yeast, bacteria and microalgae are being used to produce high value chemicals by utilizing widely available carbon sources. However, current extraction processes of many high value products from these cells are time- and labor-consuming and require toxic chemicals. This makes the extraction processes detrimental to the environment and not economically feasible. Hence, there is a demand for the development of simple, effective, and environmentally friendly method for the extraction of high value chemicals from these cell factories. Herein, we hypothesized that atomically thin edges of graphene having ability to interact with hydrophobic materials, could be used to extract high value lipids from cell factories. To achieve this, array of axially oriented graphene was deposited on iron nanoparticles. These coated nanoparticles were used to facilitate the release of intracellular lipids from Yarrowia lipolytica cells. Our treatment process can be integrated with the growth procedure and achieved the release of 50% of total cellular lipids from Y. lipolytica cells. Based on this result, we propose that nanoparticles coated with axially oriented graphene could pave efficient, environmentally friendly, and cost-effective way to release intracellular lipids from yeast cell factories.

Impact of overproduced heterologous protein characteristics on physiological response in Yarrowia lipolytica steady-state-maintained continuous cultures

Overproduction of recombinant secretory proteins triggers numerous physiological perturbations. Depending on a given heterologous protein characteristics, the producer cell is faced with different challenges which lead to varying responses in terms of its physiology and the target protein production rate. In the present study, we used steady-state-maintained Yarrowia lipolytica cells to investigate the impact of different heterologous proteins on the physiological behavior of the host cells. Such an approach allowed to uncouple the impact of the overproduction of a particular protein from the phenomena that result from growth phase or are caused by the heterogeneity of the analyzed populations. Altogether, eight variants of recombinant strains, individually overproducing heterologous proteins of varying molecular weight (27–65 kDa) and reporting activity (enzymatic and fluorescent) were subjected to chemostat cultivations. The steady-state-maintained cells were analyzed in terms of the substrate utilization, biomass and metabolites production, as well as the reporter protein synthesis. Simplified distribution of carbon and nitrogen between the respective products, as well as expression analysis of the heterologous genes were conducted. The here-obtained data suggest that using a more transcriptionally active promoter results in channeling more C flux towards the target protein, giving significantly higher specific amounts and production rates of the target polypeptide, at the cost of biomass accumulation, and with no significant impact on the polyols production. The extent of the reporter protein’s post-translational modifications, i.e., the number of disulfide bonds and glycosylation pattern, strongly impacts the synthesis process. Specific responses in terms of the protein formation kinetics, the gene expression levels, and transcript-to-protein linearity were observed.Key Points• Eight expression systems, producing different reporter proteins were analyzed.• The cells were maintained in steady-state by continuous chemostat culturing.• Protein- and promoter-specific effects were observed.

Catalytic and physical features of a naturally immobilized Yarrowia lipolytica lipase in cell debris (LipImDebri) displaying high thermostability.

Lipase activity (337U/g dry weight of cell debris) was detected in cell debris after ultrasound treatment of Yarrowia lipolytica cells cultivated in residual frying palm oil. It is a naturally immobilized lipase with protein content of 47%, herein called LipImDebri. This immobilized biocatalyst presents low hydrophobicity (8%), that can be increased adjusting pH and buffer type. Despite apparent intact cells, electron microscopy showed a shapeless and flat surface for LipImDebri and optical microscopy revealed no cell viability. Besides, an inferior mean diameter (3.4mm) in relation to whole cells reveals structure modification. A high negative zeta potential value (-33.86mV) for pH 6 and 25°C suggests that LipImDebri is a stable suspension in aqueous solution. Fourier Transform Infrared Spectra (FTIR) expose differences between LipImDebri and extracellular lipase extract signaling a physical interaction between enzyme and cell debris, which is possibly the reason for the high thermostability (k d = 0.246h-1; t 1/2 = 2.82h at 50°C, pH 7.0). A good adjustment of LipImDebri kinetic data with Hill equation (R 2 = 0.95) exposes an allosteric behavior related to the presence of more than one lipase isoform. These features reveal that LipImDebri can be a good catalyst for industrial applications.

Metabolic engineering of β-carotene biosynthesis in Yarrowia lipolytica.

Carotenoids, as potent antioxidant compounds, have gained extensive attention, especially in human health. In this study, the combination of CRISPR/Cas9 integration strategy and fermenter cultivation was utilized to obtain efficient β-carotene-producing Yarrowia lipolytica cell factories for potential industrial application. The introduction of the genes of Mucor circinelloides, encoding phytoene dehydrogenase (carB) and bifunctional phytoene synthase/lycopene cyclase (carRP), contributed to the heterologous production of β-carotene in Y. lipolytica XK2. Furthermore, β-carotene production was efficiently enhanced by increasing the copy numbers of the carB and carRP genes and overexpressing of GGS1, ERG13, and HMG, the genes related to the mevalonate (MVA) pathway. Thus, the optimized strain overexpressed a total of eight genes, including three copies of carRP, two copies of carB, and single copies of GGS1, HMG, and ERG13. As a consequence, strain Y. lipolytica XK19 accumulated approximately 408mg/L β-carotene in shake flask cultures, a twenty-four-fold increase compared to the parental strain Y. lipolytica XK2. 4.5g/L β-carotene was obtained in a 5-L fermenter through a combination of genetic engineering and culture optimization, suggesting a great capacity and flexibility of Y. lipolytica in the production of carotenoids.

Bacterial Cellulose-Alginate Composite Beads as Yarrowia lipolytica Cell Carriers for Lactone Production.

The demand for natural lactone gamma-decalactone (GDL) has increased in the fields of food and cosmetic products. However, low productivity during bioprocessing limits its industrial production. In this study, a novel composite porous cell carrier, bacterial cellulose-alginate (BC-ALG), was used for long-term biotransformation and production of GDL. The effects of this carrier on biotransformation and related mechanisms were investigated. BC-ALG carriers showed improved mechanical strength over ALG carriers, with their internal embedded cell pattern changed to an interconnected porous structure. In five repeated-batch biotransformation experiments, the maximum concentration of GDL obtained in culture with BC-ALG carriers was 8.37 g/L, approximately 3.7 times higher than that from the medium with an ALG carrier alone. The result indicated that multiple hydrogen bonding interactions at the interface between BC and ALG contributed to the compatibility and stability of BC-ALG carriers. On the basis of the above results, the BC-ALG composite carrier can be considered ideal for immobilisation of cells for the production of GDL on an industrial scale, and has the potential to be utilised in other biological processes.

Интенсивность свободнорадикального окисления в клетках Yarrowia lipolytica при выращивании в условиях экстремальных значений рН среды

Интенсивность свободнорадикального окисления в клетках Yarrowia lipolytica при выращивании в условиях экстремальных значений рН среды

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 (5mM) showed a cytostatic effect on all strains, but after 72h 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.

Use of Yarrowia lipolytica Lipase Immobilized in Cell Debris for the Production of Lipolyzed Milk Fat (LMF).

Lipase immobilized on Yarrowia lipolytica cell debris after sonication of yeast cells (LipImDebri) was used in hydrolysis reaction as a novel strategy to produce lipolyzed milk fat (LMF). Extracellular (4732.1 U/L), intracellular (130.0 U/g), and cell debris (181.0 U/g) lipases were obtained in a 4 L bioreactor using residual frying oil as inducer in 24 h fermentation process. LipImDebri showed a good operational stability retaining 70% of lipolytic activity after the second cycle and 40% after the fourth. The highest degree of hydrolysis (28%) was obtained with 500 mg LipImDebri for 6 h of lipolysis of anhydrous milk fat. LMF produced with LipImDebri presented high contents of oleic (35.2%), palmitic (25.0%), and stearic (15.4%) acids and considerable amounts of odor-active short and medium chain fatty acids (C:4–C:10) (8.13%).

Strategies to improve carotene entry into cells of Yarrowia lipolytica in a goal of encapsulation

Yeasts are good materials for encapsulation of actives, but, as they are preformed capsules, they have to be loaded. Our goal was to load cells with a big and very hydrophobic molecule, β-carotene (LogP ≈15). In classical conditions, the entry of this compound into cells is negligible and we have worked on ways to favor the entry through the cell wall. Carotene was prepared at 0.3–3 g L−1 in solvents to improve solubility and alter the cell wall structure. After a 3 h-incubation of cells with this solution, with the apolar hexane, the entry was increased to 95 μg g−1 cell wet weight but the monopolar chloroform was much more efficient (220 μg g−1 cell wet weight). However, increasing the carotene concentration in chloroform increased the adhesion of carotene on the cell wall (over 900 μg g−1 cell wet weight) but did not increase the amount into the cell. The use of ultrasound (6-min-treatment at 50% and an output control of 4) as a green physical treatment increased significantly the result for β-carotene encapsulation (852 μg g−1 cell wet weight).

On the conceptual design of a partitioning technology for the bioconversion of (+)-valencene to (+)-nootkatone on whole cells: Experimentation and modelling

The conceptual design of a four-phase partitioning bioreactor to produce (+)-nootkatone out of (+)-valencene on whole cells of Yarrowia lipolytica 2.2ab requires yet from the proper modelling and analysis of those phenomena limiting the bioconversion in this reaction system. This work is aimed at modelling this partitioning bioreactor. In this sense, a pseudo heterogeneous model is, particularly, developed by using experiments under biotic and abiotic conditions. First, in a two-phase abiotic reactor, interfacial mass transfer in absence of reaction is characterized by determining the effective interfacial coefficients for sequiterpenes (kiAdp) and for oxygen (KLa). Then, kinetics and cell deactivation are characterized by developing the corresponding models in a three-phase biotic reactor; two types of whole cells are evaluated here, namely Yarrowia lipolytica 2.2ab untreated and treated with cetyl trimethylammonium bromite (CTAB) to expel possible reaction intermediate out of the cell and with niacin (vitamin B3) to favour the regeneration of the cofactor nicotinamide adenine dinucleotide phosphate (NADPH+). It is worth stressing that kinetic and cell deactivation models account for (+)-valencene inhibition, (+)-nootkatone inhibition and oxygen inactivation. To this end, the partitioning four-phase reactor model, making use of the calculated mass transport, kinetic and deactivation parameters, is able to describe observations and predict enzyme coverage profiles at different cell activity scenarios. A parametric sensitivity study allows the identification of volume disperse phase fraction (ϕ=0.2–0.5), agitation rate (200–400rpm) and airflow rate (0.5–1vvm) as the main operational variables influencing the biological conversion of (+)-valencene. At studied conditions, the highest production rate of (+)-nootkatone is obtained at 300rpm, 1vvm and, essentially, at ϕ=0.35.

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

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