Biosurfactants production by marine yeasts isolated from zoanthids and characterization of an emulsifier produced by Yarrowia lipolytica LMS 24B.

The present study investigates the potential for biosurfactant production of 19 marine yeast species obtained from zoanthids. Using the emulsification index test to screen the samples produced by the marine yeasts, we verified that five isolates exhibited an emulsification index ≥50%. Additional tests were performed on such isolates, including oil displacement, drop collapse, Parafilm M assay, and surface tension measurement. The tolerance of produced biosurfactants for environmental conditions was also analyzed, especially considering the media's temperature, pH, and salinity. Moreover, the surfactant's ability to emulsify different hydrocarbon sources and to metabolize kerosene as the sole carbon source was evaluated in vitro. Our results demonstrate that yeast biosurfactants can emulsify hydrocarbon sources under different physicochemical conditions and metabolize kerosene as a carbon source. Considering the Yarrowia lipolytica LMS 24B as the yeast model for biosurfactant production from the cell's wall biomass, emulsification indexes of 61.2% were obtained, even at a high temperature of 120 °C. Furthermore, the Fourier-transform middle infrared spectroscopy (FTIR) analysis of the biosurfactant's chemical composition revealed the presence of distinct functional groups assigned to a glycoprotein complex. Considering the status of developing new bioproducts and bioprocesses nowadays, our findings bring a new perspective to biosurfactant production by marine yeasts, especially Y. lipolytica LMS 24B. In particular, the presented results validate the relevance of marine environments as valuable sources of genetic resources, i.e., yeast strains capable of metabolizing and emulsifying petroleum derivatives.

Engineering Yarrowia lipolytica for Sustainable Production of the Pomegranate Seed Oil-Derived Punicic Acid.

Punicic acid is a conjugated linolenic acid with various biological activities including antiobesity, antioxidant, anticancer, and anti-inflammatory effects. It is often used as a nutraceutical, dietary additive, and animal feed. Currently, punicic acid is primarily extracted from pomegranate seed oil, but it is restricted due to the extended growth cycle, climatic limitations, and low recovery level. There have also been reports on the chemical synthesis of punicic acid, but it resulted in a mixture of structurally similar isomers, requiring additional purification/separation steps. In this study, a comprehensive strategy for the production of punicic acid in Yarrowia lipolytica was implemented by pushing the supply of linoleic acid precursors in a high-oleic oil strain, expressing multiple copies of the fatty acid conjugase gene from Punica granatum, engineering the acyl-editing pathway to improve the phosphatidylcholine pool, and promoting the assembly of punicic acid in the form of triglycerides. The optimal strain with high oil production capacity and a significantly increased punicic acid ratio accumulated 3072.72 mg/L punicic acid, accounting for 6.19% of total fatty acids in fed-batch fermentation, providing a viable, sustainable, and green approach for punicic acid production to substitute plant extraction and chemical synthesis production.

Exploring the use of hexadecane by Yarrowia lipolytica: Effect of dissolved oxygen and medium supplementation.

This work aimed to evaluate the effect of medium composition and volumetric oxygen transfer coefficient (kLa) on Y. lipolytica growth and production of microbial lipids and enzymes from hexadecane. In the stirred tank bioreactor, increasing kLa from 11 h-1 to 132 h-1 improved the hexadecane assimilation rate, biomass concentration, and lipids synthesis (0.90 g·L-1). A cost-effective hexadecane-based medium supplemented with corn steep liquor and a low amount of ammonium sulfate boosted lipids production up to 2.1 g·L-1, composed of palmitic, palmitoleic, oleic, and linoleic acids. The unsaturated/saturated fraction was dependent on the C/N ratio. Lipids of Y. lipolytica CBS 2075 are promising feedstock for animal feed, food additives, or the biodiesel industry. Simultaneous synthesis of extracellular lipase and protease from hexadecane was observed, which is a new feature that was not previously reported. The highest enzyme activity was obtained at the highest C/N ratio conditions. These results open new perspectives on the application of Y. lipolytica-based cultures for the biotransformation of hexadecane-polluted streams into valuable compounds, fulfilling an interesting strategy towards the circular economy concept.

Revealing the Mechanisms of Enhanced ß-Farnesene Production in Yarrowia lipolytica through Metabolomics Analysis.

ß-Farnesene is an advanced molecule with promising applications in agriculture, the cosmetics industry, pharmaceuticals, and bioenergy. To supplement the shortcomings of rational design in the development of high-producing ß-farnesene strains, a Metabolic Pathway Design-Fermentation Test-Metabolomic Analysis-Target Mining experimental cycle was designed. In this study, by over-adding 20 different amino acids/nucleobases to induce fluctuations in the production of ß-farnesene, the changes in intracellular metabolites in the ß-farnesene titer-increased group were analyzed using non-targeted metabolomics. Differential metabolites that were detected in each experimental group were selected, and their metabolic pathways were located. Based on these differential metabolites, targeted strain gene editing and culture medium optimization were performed. The overexpression of the coenzyme A synthesis-related gene pantothenate kinase (PanK) and the addition of four mixed water-soluble vitamins in the culture medium increased the ß-farnesene titer in the shake flask to 1054.8 mg/L, a 48.5% increase from the initial strain. In the subsequent fed-batch fermentation, the ß-farnesene titer further reached 24.6 g/L. This work demonstrates the tremendous application value of metabolomics analysis for the development of industrial recombinant strains and the optimization of fermentation conditions.

Beet red food colourant can be produced more sustainably with engineered Yarrowia lipolytica.

Synthetic food colourants are widely used in the food industry, but consumer concerns about safety and sustainability are driving a need for natural food-colour alternatives. Betanin, which is extracted from red beetroots, is a commonly used natural red food colour. However, the betanin content of beetroot is very low (~0.2% wet weight), which means that the extraction of betanin is incredibly wasteful in terms of land use, processing costs and vegetable waste. Here we developed a sustainability-driven biotechnological process for producing red beet betalains, namely, betanin and its isomer isobetanin, by engineering the oleaginous yeast Yarrowia lipolytica. Metabolic engineering and fermentation optimization enabled production of 1,271 ± 141 mg l-1 betanin and 55 ± 7 mg l-1 isobetanin in 51 h using glucose as carbon source in controlled fed-batch fermentations. According to a life cycle assessment, at industrial scale (550 t yr-1), our fermentation process would require significantly less land, energy and resources compared with the traditional extraction of betanin from beetroot crops. Finally, we apply techno-economic assessment to show that betanin production by fermentation could be economically feasible in the existing market conditions.

Effect of Yarrowia lipolytica yeast biomass with increased kynurenic acid content on selected metabolic indicators in mice.

Background: The unconventional yeast species Yarrowia lipolytica is a valuable source of protein and many other nutrients. It can be used to produce hydrolytic enzymes and metabolites, including kynurenic acid (KYNA), an endogenous metabolite of tryptophan with a multidirectional effect on the body. The administration of Y. lipolytica with an increased content of KYNA in the diet may have a beneficial effect on metabolism, which was evaluated in a nutritional experiment on mice. Methods: In the dry biomass of Y. lipolytica S12 enriched in KYNA (high-KYNA yeast) and low-KYNA (control) yeast, the content of KYNA was determined by high-performance liquid chromatography. Then, proximate and amino acid composition and selected indicators of antioxidant status were compared. The effect of 5% high-KYNA yeast content in the diet on the growth, hematological and biochemical indices of blood and the redox status of the liver was determined in a 7-week experiment on adult male mice from an outbred colony derived from A/St, BALB/c, BN/a and C57BL/6J inbred strains. Results: High-KYNA yeast was characterized by a greater concentration of KYNA than low-KYNA yeast (0.80 ± 0.08 vs. 0.29 ± 0.01 g/kg dry matter), lower content of crude protein with a less favorable amino acid composition and minerals, higher level of crude fiber and fat and lower ferric-reducing antioxidant power, concentration of phenols and glutathione. Consumption of the high-KYNA yeast diet did not affect the cumulative body weight gain per cage, cumulative food intake per cage and protein efficiency ratio compared to the control diet. A trend towards lower mean corpuscular volume and hematocrit, higher mean corpuscular hemoglobin concentration and lower serum total protein and globulins was observed, increased serum total cholesterol and urea were noted. Its ingestion resulted in a trend towards greater ferric-reducing antioxidant power in the liver and did not affect the degree of liver lipid and protein oxidation. Conclusions: The improvement of the quality of Y. lipolytica yeast biomass with increased content of KYNA, including its antioxidant potential, would be affected by the preserved level of protein and unchanged amino acid profile. It will be worth investigating the effect of such optimized yeast on model animals, including animals with metabolic diseases.

The Effect of Plasma-Treated Water on Microbial Growth and Biosynthesis of Gamma-Decalactones by Yarrowia lipolytica Yeast.

In recent years, the production of plasma-treated water (PTW) by low-temperature low-pressure glow plasma (LPGP) has been increasingly gaining in popularity. LPGP-treated water changes its physical and physiochemical properties compared to standard distilled water. In this study, a non-conventional lipolytic yeast species Yarrowia lipolytica was cultivated in culture media based on Nantes plasma water with heightened singlet oxygen content (Nantes PW) or in water treated with low-temperature, low-pressure glow plasma while in contact with air (PWTA) or nitrogen (PWTN). The research aimed to assess the influence of culture conditions on castor oil biotransformation to gamma-decalactone (GDL) and other secondary metabolites in media based on nanowater. The Nantes plasma water-based medium attained the highest concentration of gamma-decalactone (4.81 ± 0.51 g/L at 144 h of culture), maximum biomass concentration and biomass yield from the substrate. The amplified activity of lipases in the nanowater-based medium, in comparison to the control medium, is encouraging from the perspective of GDL biosynthesis, relying on the biotransformation of ricinoleic acid, which is the primary component of castor oil. Although lipid hydrolysis was enhanced, this step seemed not crucial for GDL concentration. Interestingly, the study validates the significance of oxygen in ß-oxidation enzymes and its role in the bioconversion of ricinoleic acid to GDL and other lactones. Specifically, media with higher oxygen content (WPTA) and Nantes plasma water resulted in remarkably high concentrations of four lactones: gamma-decalactone, 3-hydroxy-gamma-decalactone, dec-2-en-4-olide and dec-3-en-4-olide.

Combination of microbial and chemical synthesis for the sustainable production of ß-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 ß-elemene production. In this study, we designed a Yarrowia lipolytica cell factory that can continuously produce germacrene A, which is further converted into ß-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 ß-elemene titer reported to date with 5.08 g/L. This sustainable and green method has the potential for industrial ß-elemene production.

Simultaneous bioreduction of tellurite and selenite by Yarrowia lipolytica, Trichosporon cutaneum, and their co-culture along with characterization of biosynthesized Te-Se nanoparticles.

BACKGROUND: Natural and anthropogenic activities, such as weathering of rocks and industrial processes, result in the release of toxic oxyanions such as selenium (Se) and tellurium (Te) into the environment. Due to the high toxicity of these compounds, their removal from the environment is vital. RESULTS: In this study, two yeast strains, Yarrowia lipolytica and Trichosporon cutaneum, were selected as the superior strains for the bioremediation of tellurium and selenium. The reduction analyses showed that exposure to selenite induced more detrimental effects on the strains compared to tellurite. In addition, co-reduction of pollutants displayed almost the same results in selenite reduction and more than ~ 20% higher tellurite reduction in 50 h, which shows that selenite triggered higher tellurite reduction in both strains. The selenite and tellurite kinetics of removal were consistent with the first-order model because of their inhibitory behavior. The result of several characterization experiments, such as FE-SEM (Field emission scanning electron microscopy), dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR), X-ray diffractometer (XRD), and dispersive X-ray (EDX) on Te-Se nanoparticles (NPs) revealed that the separated Te-Se NPs were needle-like, spherical, and amorphous, consisted of Te-Se NPs ranging from 25 to 171 nm in size, and their surface was covered with different biomolecules. CONCLUSIONS: Remarkably, this work shows, for the first time, the simultaneous bioreduction of tellurite and selenite and the production of Te-Se NPs using yeast strains, indicating their potential in this area, which may be applied to the nanotechnology industry and environmental remediation.

Multi-omics view of recombinant Yarrowia lipolytica: Enhanced ketogenic amino acid catabolism increases polyketide-synthase-driven docosahexaenoic production to high selectivity at the gram scale.

DHA is a marine PUFA of commercial value, given its multiple health benefits. The worldwide emerging shortage in DHA supply has increased interest in microbial cell factories that can provide the compound de novo. In this regard, the present work aimed to improve DHA production in the oleaginous yeast strain Y. lipolytica Af4, which synthetized the PUFA via a heterologous myxobacterial polyketide synthase (PKS)-like gene cluster. As starting point, we used transcriptomics, metabolomics, and 13C-based metabolic pathway profiling to study the cellular dynamics of Y. lipolytica Af4. The shift from the growth to the stationary DHA-production phase was associated with fundamental changes in carbon core metabolism, including a strong upregulation of the PUFA gene cluster, as well as an increase in citrate and fatty acid degradation. At the same time, the intracellular levels of the two DHA precursors acetyl-CoA and malonyl-CoA dropped by up to 98% into the picomolar range. Interestingly, the degradation pathways for the ketogenic amino acids l-lysine, l-leucine, and l-isoleucine were transcriptionally activated, presumably to provide extra acetyl-CoA. Supplementation with small amounts of these amino acids at the beginning of the DHA production phase beneficially increased the intracellular CoA-ester pools and boosted the DHA titer by almost 40%. Isotopic 13C-tracer studies revealed that the supplements were efficiently directed toward intracellular CoA-esters and DHA. Hereby, l-lysine was found to be most efficient, as it enabled long-term activation, due to storage within the vacuole and continuous breakdown. The novel strategy enabled DHA production in Y. lipolytica at the gram scale for the first time. DHA was produced at a high selectivity (27% of total fatty acids) and free of the structurally similar PUFA DPA, which facilitates purification for high-value medical applications that require API-grade DHA. The assembled multi-omics picture of the central metabolism of Y. lipolytica provides valuable insights into this important yeast. Beyond our work, the enhanced catabolism of ketogenic amino acids seems promising for the overproduction of other compounds in Y. lipolytica, whose synthesis is limited by the availability of CoA ester precursors.

Single cell protein and oil production from solid cocoa fatty acid distillates co-fed ethanol.

The use of solid lipid sidestreams have been overlooked as a feedstock for the production of microbial biomass for food and feed applications and little to no recent work has examined the utilization of solid fatty acid distillates (FADs), which are a significant residue from vegetable oil processing. Yarrowia lipolytica and Rhodosporidium toruloides cultivated on cocoa fatty acid distillates (CFAD) generated final cell dry weight values > 40 g/L, with strong productivity (3.3 g/L·h) and rich protein (>45%) and lipid content (>25%). Interestingly, microbial oils were > 65% unsaturated fatty acids, compared < 20% unsaturated content in FAD. Importantly, to overcome mass-transfer limitations associated with bioconversion of solid lipid residues, ethanol was applied as a co-substrate to solubilize FAD residues. Here, FAD residues from cocoa deodorization have been demonstrated to be high energy feedstocks that represent an attractive substrate for the production of both single cell protein and oil (SCPO).

Genetic inactivation of the Carnitine/Acetyl-Carnitine mitochondrial carrier of Yarrowia lipolytica leads to enhanced odd-chain fatty acid production.

BACKGROUND: Mitochondrial carriers (MCs) can deeply affect the intracellular flux distribution of metabolic pathways. The manipulation of their expression level, to redirect the flux toward the production of a molecule of interest, is an attractive target for the metabolic engineering of eukaryotic microorganisms. The non-conventional yeast Yarrowia lipolytica is able to use a wide range of substrates. As oleaginous yeast, it directs most of the acetyl-CoA therefrom generated towards the synthesis of lipids, which occurs in the cytoplasm. Among them, the odd-chain fatty acids (OCFAs) are promising microbial-based compounds with several applications in the medical, cosmetic, chemical and agricultural industries. RESULTS: In this study, we have identified the MC involved in the Carnitine/Acetyl-Carnitine shuttle in Y. lipolytica, YlCrc1. The Y. lipolytica Ylcrc1 knock-out strain failed to grow on ethanol, acetate and oleic acid, demonstrating the fundamental role of this MC in the transport of acetyl-CoA from peroxisomes and cytoplasm into mitochondria. A metabolic engineering strategy involving the deletion of YlCRC1, and the recombinant expression of propionyl-CoA transferase from Ralstonia eutropha (RePCT), improved propionate utilization and its conversion into OCFAs. These genetic modifications and a lipogenic medium supplemented with glucose and propionate as the sole carbon sources, led to enhanced accumulation of OCFAs in Y. lipolytica. CONCLUSIONS: The Carnitine/Acetyl-Carnitine shuttle of Y. lipolytica involving YlCrc1, is the sole pathway for transporting peroxisomal or cytosolic acetyl-CoA to mitochondria. Manipulation of this carrier can be a promising target for metabolic engineering approaches involving cytosolic acetyl-CoA, as demonstrated by the effect of YlCRC1 deletion on OCFAs synthesis.

Yarrowia lipolytica as a Platform for Punicic Acid Production.

Punicic acid (PuA) is a polyunsaturated fatty acid with significant medical, biological, and nutraceutical properties. The primary source of punicic acid is the pomegranate seed oil obtained from fruits of trees that are mainly cultivated in subtropical and tropical climates. To establish sustainable production of PuA, various recombinant microorganisms and plants have been explored as platforms with limited efficiencies. In this study, the oleaginous yeast Yarrowia lipolytica was employed as a host for PuA production. First, growth and lipid accumulation of Y. lipolytica were evaluated in medium supplemented with pomegranate seed oil, resulting in the accumulation of lipids up to 31.2%, consisting of 22% PuA esterified in the fraction of glycerolipids. In addition, lipid-engineered Y. lipolytica strains, transformed with the bifunctional fatty acid conjugase/desaturase from Punica granatum (PgFADX), showed the ability to accumulate PuA de novo. PuA was detected in both polar and neutral lipid fractions, especially in phosphatidylcholine and triacylglycerols. Promoter optimization for PgFADX expression resulted in improved accumulation of PuA from 0.9 to 1.8 mg/g of dry cell weight. The best-producing strain expressing PgFADX under the control of a strong erythritol-inducible promoter produced 36.6 mg/L PuA. These results demonstrate that the yeast Y. lipolytica is a promising host for PuA production.

Combining orthogonal plant and non-plant fatty acid biosynthesis pathways for efficient production of microbial oil enriched in nervonic acid in Yarrowia lipolytica.

Nervonic acid has proven efficacy in brain development and the prevention of neurodegenerative diseases. Here, an alternative and sustainable strategy for nervonic acid-enriched plant oil production was established. Different ß-ketoacyl-CoA synthases and heterologous Δ15 desaturase were co-expressed, combined with the deletion of the ß-oxidation pathway to construct orthogonal plant and non-plant nervonic acid biosynthesis pathways in Yarrowia lipolytica. A "block-pull-restrain" strategy was further applied to improve the supply of stearic acid as the precursor of the non-plant pathway. Then, lysophosphatidic acid acyltransferase from Malania oleifera (MoLpaat) was identified, which showed specificity for nervonic acid. Endogenous LPAAT was exchanged by MoLPAAT resulted in 17.10 % nervonic acid accumulation. Finally, lipid metabolism was engineered and cofactor supply was increased to boost the lipid accumulation in a stable null-hyphal strain. The final strain produced 57.84 g/L oils with 23.44 % nervonic acid in fed-batch fermentation, which has the potential to substitute nervonic acid-enriched plant oil.

Isolation, purification, and mass spectrometry identification of the enzyme involved in citrus flavor (+)-valencene biotransformation to (+)-nootkatone by Yarrowia lipolytica.

BACKGROUND: (+)-Nootkatone is a highly valuable sesquiterpene compound that can be used as an aromatic in the food industry because of its grapefruit flavor and low sensory threshold. The unconventional yeast Yarrowia lipolytica has many unique physical and chemical properties, metabolic characteristics, and genetic structure, which has aroused the interest of researchers. Previous research showed that Y. lipolytica possesses the ability to transform the sesquiterpene (+)-valencene to (+)-nootkatone. The aim of this study was to isolate, purify, and identify the enzyme involved in the (+)-valencene bioconversion to (+)-nootkatone by Y. lipolytica. RESULTS: In this study, ultrasonic-assisted extraction, ammonium sulfate precipitation, anion-exchange chromatography, and gel-filtration chromatography were used to separate and purify the enzyme involved in the (+)-valencene bioconversion by Y. lipolytica. The protein was identified as aldehyde dehydrogenase (ALDH) (gene0658) using sodium dodecyl sulfate polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis. The ALDH had the highest activity when the pH value was 6.0 and the temperature was 30 °C. The activity of ALDH was significantly stimulated by ferrous ions and inhibited by barium, calcium, and magnesium ions. CONCLUSION: This is the first time that ALDH was found to participate in (+)-valencene biotransformation by Y. lipolytica. It may be involved in regulating the microbial transformation of (+)-valencene to (+)-nootkatone through redox characteristics. This study provides a theoretical basis and reference for the biological synthesis of citrus flavor (+)-nootkatone. © 2023 Society of Chemical Industry.

Using oils and fats to replace sugars as feedstocks for biomanufacturing: Challenges and opportunities for the yeast Yarrowia lipolytica.

More than 200 million tons of plant oils and animal fats are produced annually worldwide from oil, crops, and the rendered animal fat industry. Triacylglycerol, an abundant energy-dense compound, is the major form of lipid in oils and fats. While oils or fats are very important raw materials and functional ingredients for food or related products, a significant portion is currently diverted to or recovered as waste. To significantly increase the value of waste oils or fats and expand their applications with a minimal environmental footprint, microbial biomanufacturing is presented as an effective strategy for adding value. Though both bacteria and yeast can be engineered to use oils or fats as the biomanufacturing feedstocks, the yeast Yarrowia lipolytica is presented as one of the most attractive platforms. Y. lipolytica is oleaginous, generally regarded as safe, demonstrated as a promising industrial producer, and has unique capabilities for efficient catabolism and bioconversion of lipid substrates. This review summarizes the major challenges and opportunities for Y. lipolytica as a new biomanufacturing platform for the production of value-added products from oils and fats. This review also discusses relevant cellular and metabolic engineering strategies such as fatty acid transport, fatty acid catabolism and bioconversion, redox balances and energy yield, cell morphology and stress response, and bioreaction engineering. Finally, this review highlights specific product classes including long-chain diacids, wax esters, terpenes, and carotenoids with unique synthesis opportunities from oils and fats in Y. lipolytica.

Tailoring and optimizing fatty acid production by oleaginous yeasts through the systematic exploration of their physiological fitness.

BACKGROUND: The use of palm oil for our current needs is unsustainable. Replacing palm oil with oils produced by microbes through the conversion of sustainable feedstocks is a promising alternative. However, there are major technical challenges that must be overcome to enable this transition. Foremost among these challenges is the stark increase in lipid accumulation and production of higher content of specific fatty acids. Therefore, there is a need for more in-depth knowledge and systematic exploration of the oil productivity of the oleaginous yeasts. In this study, we cultivated Cutaneotrichosporon oleaginosus and Yarrowia lipolytica at various C/N ratios and temperatures in a defined medium with glycerol as carbon source and urea as nitrogen source. We ascertained the synergistic effect between various C/N ratios of a defined medium at different temperatures with Response Surface Methodology (RSM) and explored the variation in fatty acid composition through Principal Component Analysis. RESULTS: By applying RSM, we determined a temperature of 30 °C and a C/N ratio of 175 g/g to enable maximal oil production by C. oleaginosus and a temperature of 21 °C and a C/N ratio of 140 g/g for Y. lipolytica. We increased production by 71% and 66% respectively for each yeast compared to the average lipid accumulation in all tested conditions. Modulating temperature enabled us to steer the fatty acid compositions. Accordingly, switching from higher temperature to lower cultivation temperature shifted the production of oils from more saturated to unsaturated by 14% in C. oleaginosus and 31% in Y. lipolytica. Higher cultivation temperatures resulted in production of even longer saturated fatty acids, 3% in C. oleaginosus and 1.5% in Y. lipolytica. CONCLUSIONS: In this study, we provided the optimum C/N ratio and temperature for C. oleaginosus and Y. lipolytica by RSM. Additionally, we demonstrated that lipid accumulation of both oleaginous yeasts was significantly affected by the C/N ratio and temperature. Furthermore, we systematically analyzed the variation in fatty acids composition and proved that changing the C/N ratio and temperature steer the composition. We have further established these oleaginous yeasts as platforms for production of tailored fatty acids.

Enhancing very long chain fatty acids production in Yarrowia lipolytica.

BACKGROUND: Very long chain fatty acids (VLCFA) and their derivatives are industrially attractive compounds. The most important are behenic acid (C22:0) and erucic acid (C22:1Δ13), which are used as lubricants, and moisturizers. C22:0 and C22:1Δ13 have also potential for biofuel production. These fatty acids are conventionally obtained from plant oils. Yarrowia lipolytica is an oleaginous yeast with a long history of gene manipulations resulting in the production of industrially interesting compounds, such as organic acids, proteins, and various lipophilic molecules. It has been shown previously that it has potential for the production of VLCFA enriched single cell oils. RESULTS: The metabolism of Y. lipolytica was redesigned to achieve increased production of VLCFA. The effect of native diacylglycerol acyltransferases of this yeast YlLro1p, YlDga1p, and YlDga2p on the accumulation of VLCFA was examined. It was found that YlDga1p is the only enzyme with a beneficial effect. Further improvement of accumulation was achieved by overexpression of 3-ketoacyl-CoA synthase (TaFAE1) under 8UAS-pTEF promoter and blockage fatty acid degradation pathway by deletion of YlMFE1. The best-producing strain YL53 (Δmfe, pTEF-YlDGA1, 8UAS-pTEF-TaFAE1) produced 120 µg of very long chain fatty acids per g of produced biomass, which accounted for 34% of total fatty acids in biomass. CONCLUSIONS: Recombinant strains of Y. lipolytica have proved to be good producers of VLCFA. Redesign of lipid metabolism pathways had a positive effect on the accumulation of C22:1Δ13 and C22:0, which are technologically attractive compounds.

Yarrowia lipolytica as an Alternative and Valuable Source of Nutritional and Bioactive Compounds for Humans.

Yarrowia lipolytica, an oleagineous species of yeast, is a carrier of various important nutrients. The biomass of this yeast is an extensive source of protein, exogenous amino acids, bioavailable essenctial trace minerals, and lipid compounds as mainly unsaturated fatty acids. The biomass also contains B vitamins, including vitamin B12, and many other bioactive components. Therefore, Y. lipolytica biomass can be used in food supplements for humans as safe and nutritional additives for maintaining the homeostasis of the organism, including for vegans and vegetarians, athletes, people after recovery, and people at risk of B vitamin deficiencies.

Bio-oil production for biodiesel industry by Yarrowia lipolytica from volatile fatty acids in two-stage batch culture.

Microbial lipids-derived biodiesel is garnering much attention owing to its potential to substitute diesel fuel. In this study, lipid accumulation by Yarrowia lipolytica from volatile fatty acids (VFAs) was studied in a lab-scale stirred tank bioreactor. In batch cultures, Y. lipolytica NCYC 2904 was able to grow in 18 g·L-1 of VFAs (acetate, propionate, and butyrate), and the addition of a co-substrate (glucose) led to a fivefold improvement in lipid concentration. Furthermore, the two-stage batch culture (growth phase in glucose (1st stage) followed by a lipogenic phase in VFAs (2nd stage)) was the best strategy to obtain the highest lipid content in the cells (37%, w/w), with aeration conditions that kept dissolved oxygen concentration between 40% and 50% of saturation during the lipogenic phase. The estimated fuel properties of biodiesel produced from Y. lipolytica NCYC 2904 lipids are comparable with those of the biodiesel produced from vegetable oils and are in accordance with the international standards (EN 14214 and ASTM D6751). The cultivation strategies herein devised enable a sustainable, eco-friendly, and economical production of microbial lipids, based on feedstocks such as VFAs that can be derived from the acidogenic fermentation of organic wastes. KEY POINTS: • Addition of glucose to VFAs enhances lipids in Y. lipolytica in batch cultures • Two-stage batch culture - growth in glucose followed by VFAs pulse - rises lipids • Dissolved oxygen of 40-50% of saturation is crucial at the lipogenic phase.