Yarrowia Lipolytica Strains Research Articles

Production of human milk fat substitute by engineered strains of Yarrowia lipolytica

Human milk fat has a distinctive stereoisomeric structure where palmitic acid is esterified to the middle (sn-2) position on the glycerol backbone of the triacylglycerol and unsaturated fatty acids to the outer (sn-1/3) positions. This configuration allows for more efficient nutrient absorption in the infant gut. However, the fat used in most infant formulas originates from plants, which exclude palmitic acid from the sn-2 position. Oleaginous yeasts provide an alternative source of lipids for human nutrition. However, these yeasts also exclude palmitic acid from the sn-2 position of their triacylglycerol. Here we show that Yarrowia lipolytica can be engineered to produce triacylglycerol with more than 60% of the palmitic acid in the sn-2 position, by expression of lysophosphatidic acid acyltransferases with palmitoyl-Coenzyme A specificity. The engineered Y. lipolytica strains can be cultured on glycerol, glucose, palm oil or a mixture of substrates, under nitrogen limited condition, to produce triacylglycerol with a fatty acid composition that resembles human milk fat, in terms of the major molecular species (palmitic, oleic and linoleic acids). Culture on palm oil or a mixture of glucose and palm oil produced the highest lipid titre and a triacylglycerol composition that is most similar with human milk fat. Our data show that an oleaginous yeast can be engineered to produce a human milk fat substitute (β-palmitate), that could be used as an ingredient in infant formulas.

Hyperosmolarity adversely impacts recombinant protein synthesis by Yarrowia lipolytica\u2014molecular background revealed by quantitative proteomics

In this research, we were interested in answering a question whether subjecting a Yarrowia lipolytica strain overproducing a recombinant secretory protein (rs-Prot) to pre-optimized stress factors may enhance synthesis of the rs-Prot. Increased osmolarity (3 Osm kg−1) was the primary stress factor implemented alone or in combination with decreased temperature (20 °C), known to promote synthesis of rs-Prots. The treatments were executed in batch bioreactor cultures, and the cellular response was studied in terms of culture progression, gene expression and global proteomics, to get insight into molecular bases underlying an awaken reaction. Primarily, we observed that hyperosmolarity executed by high sorbitol concentration does not enhance synthesis of the rs-Prot but increases its transcription. Expectedly, hyperosmolarity induced synthesis of polyols at the expense of citric acid synthesis and growth, which was severely limited. A number of stress-related proteins were upregulated, including heat-shock proteins (HSPs) and aldo–keto reductases, as observed at transcriptomics and proteomics levels. Concerted downregulation of central carbon metabolism, including glycolysis, tricarboxylic acid cycle and fatty acid synthesis, highlighted redirection of carbon fluxes. Elevated abundance of HSPs and osmolytes did not outbalance the severe limitation of protein synthesis, marked by orchestrated downregulation of translation (elongation factors, several aa-tRNA synthetases), amino acid biosynthesis and ribosome biogenesis in response to the hyperosmolarity. Altogether we settled that increased osmolarity is not beneficial for rs-Prots synthesis in Y. lipolytica, even though some elements of the response could assist this process. Insight into global changes in the yeast proteome under the treatments is provided.Key points• Temp enhances, but Osm decreases rs-Prots synthesis by Y. lipolytica.• Enhanced abundance of HSPs and osmolytes is overweighted by limited translation.• Global proteome under Osm, Temp and Osm Temp treatments was studied.

Bioconversions of Biodiesel-Derived Glycerol into Sugar Alcohols by Newly Isolated Wild-Type Yarrowia lipolytica Strains

The utilization of crude glycerol, generated as a by-product from the biodiesel production process, for the production of high value-added products represents an opportunity to overcome the negative impact of low glycerol prices in the biodiesel industry. In this study, the biochemical behavior of Yarrowia lipolytica strains FMCC Y-74 and FMCC Y-75 was investigated using glycerol as a carbon source. Initially, the effect of pH value (3.0–7.0) was examined to produce polyols, intracellular lipids, and polysaccharides. At low pH values (initial pH 3.0–5.0), significant mannitol production was recorded. The highest mannitol production (19.64 g L−1) was obtained by Y. lipolytica FMCC Y-74 at pH = 3.0. At pH values ranging between 5.0 and 6.0, intracellular polysaccharides synthesis was favored, while polyols production was suppressed. Subsequently, the effect of crude glycerol and its concentration on polyols production was studied. Y. lipolytica FMCC Y-74 showed high tolerance to impurities of crude glycerol. Initial substrate concentrations influence polyols production and distribution with a metabolic shift toward erythritol production being observed when the initial glycerol concentration (Gly0) increased. The highest total polyols production (=56.64 g L−1) was obtained at Gly0 adjusted to ≈120 g L−1. The highest polyols conversion yield (0.59 g g−1) and productivity (4.36 g L−1 d−1) were reached at Gly0 = 80 g L−1. In fed-batch intermittent fermentation with glycerol concentration remaining ≤60 g L−1, the metabolism was shifted toward mannitol biosynthesis, which was the main polyol produced in significant quantities (=36.84 g L−1) with a corresponding conversion yield of 0.51 g g−1.

Synthesis, Biological Evaluation, and Structure-Activity Relationships of 4-Aminopiperidines as Novel Antifungal Agents Targeting Ergosterol Biosynthesis.

The aliphatic heterocycles piperidine and morpholine are core structures of well-known antifungals such as fenpropidin and fenpropimorph, commonly used as agrofungicides, and the related morpholine amorolfine is approved for the treatment of dermal mycoses in humans. Inspired by these lead structures, we describe here the synthesis and biological evaluation of 4-aminopiperidines as a novel chemotype of antifungals with remarkable antifungal activity. A library of more than 30 4-aminopiperidines was synthesized, starting from N-substituted 4-piperidone derivatives by reductive amination with appropriate amines using sodium triacetoxyborohydride. Antifungal activity was determined on the model strain Yarrowia lipolytica, and some compounds showed interesting growth-inhibiting activity. These compounds were tested on 20 clinically relevant fungal isolates (Aspergillus spp., Candida spp., Mucormycetes) by standardized microbroth dilution assays. Two of the six compounds, 1-benzyl-N-dodecylpiperidin-4-amine and N-dodecyl-1-phenethylpiperidin-4-amine, were identified as promising candidates for further development based on their in vitro antifungal activity against Candida spp. and Aspergillus spp. Antifungal activity was determined for 18 Aspergillus spp. and 19 Candida spp., and their impact on ergosterol and cholesterol biosynthesis was determined. Toxicity was determined on HL-60, HUVEC, and MCF10A cells, and in the alternative in vivo model Galleria mellonella. Analysis of sterol patterns after incubation gave valuable insights into the putative molecular mechanism of action, indicating inhibition of the enzymes sterol C14-reductase and sterol C8-isomerase in fungal ergosterol biosynthesis.

Screening of Biosurfactant Production by Yarrowia lipolytica Strains and Evaluation of Their Antibiofilm and Anti-Adhesive Activities against Salmonella enterica ser. Enteritidis Biofilms

Screening of Biosurfactant Production by Yarrowia lipolytica Strains and Evaluation of Their Antibiofilm and Anti-Adhesive Activities against Salmonella enterica ser. Enteritidis Biofilms

A comparison of the production of gamma-decalactone in wild-type and mutant strains of Yarrowia lipolytica

A comparison of the production of gamma-decalactone in wild-type and mutant strains of Yarrowia lipolytica

Recombinant Yarrowia lipolytica strains for the heterologous expression of multi-component enzyme systems: Expression of mammalian steroidogenic proteins

New Yarrowia lipolytica strains for the co-expression of steroidogenic mammalian proteins were obtained in this study. For this purpose, a two-step approach for constructing recombinant strains that permits the simple introduction of several expression cassettes encoding heterologous proteins into the yeast genome was successfully applied. This study tested two series of integrative multi-copy expression vectors containing cDNAs for the mature forms of P450scc system components (cytochrome P450scc (CYP11A1), adrenodoxin reductase, adrenodoxin, or fused adrenodoxin-P450scc) or for P45017α (CYP17A1) under the control of the isocitrate lyase promoter pICL1, which were constructed using the basic plasmids p64PT or p67PT (rDNA or the long terminal repeat (LTR) zeta of Ylt1 as integration targeting sequences and ura3d4 as a multi-copy selection marker). This study demonstrated the integration of up to three expression vectors containing different heterologous cDNA via their simultaneous transformation into haploid recipient strains. Additionally, further combinations of the different expression cassettes in one strain were obtained by subsequent diploidisation using selected haploid multi-copy transformants. Thus, recombinant strains containing three to five different expression cassettes were obtained, as demonstrated by Southern blotting. Expression of P450scc system proteins was identified by western blotting. The presented method for recombinant strain construction is a useful tool for the heterologous expression of multi-component enzyme systems in Y. lipolytica.

Yarrowia lipolytica Strains and Their Biotechnological Applications: How Natural Biodiversity and Metabolic Engineering Could Contribute to Cell Factories Improvement.

Among non-conventional yeasts of industrial interest, the dimorphic oleaginous yeast Yarrowia lipolytica appears as one of the most attractive for a large range of white biotechnology applications, from heterologous proteins secretion to cell factories process development. The past, present and potential applications of wild-type, traditionally improved or genetically modified Yarrowia lipolytica strains will be resumed, together with the wide array of molecular tools now available to genetically engineer and metabolically remodel this yeast. The present review will also provide a detailed description of Yarrowia lipolytica strains and highlight the natural biodiversity of this yeast, a subject little touched upon in most previous reviews. This work intends to fill this gap by retracing the genealogy of the main Yarrowia lipolytica strains of industrial interest, by illustrating the search for new genetic backgrounds and by providing data about the main publicly available strains in yeast collections worldwide. At last, it will focus on exemplifying how advances in engineering tools can leverage a better biotechnological exploitation of the natural biodiversity of Yarrowia lipolytica and of other yeasts from the Yarrowia clade.

Bioprocess development using organic biowaste and sustainability assessment of succinic acid production with engineered Yarrowia lipolytica strain

The utilization of crude waste streams and the development of novel bioprocessing routes are necessary in order to expand the industrial implementation of bio-based succinic acid production. This work focuses on the utilization of zero cost municipal organic biowaste for the production of bio-based succinic acid using the engineered yeast strain Yarrowia lipolytica PSA02004. Fermentation advances focused on pH regulation towards reduced NaOH requirements. Optimum initial carbon source concentration (100–120 g/L) in crude hydrolysates was estimated in shake flask cultures. Optimum kLα (183.2 h−1) in fed-batch cultures resulted in 42.2 g/L succinic acid with 0.38 g/g yield and 0.84 g/L/h productivity. A two-stage pH regulation strategy was employed to enhance succinic acid production efficiency and reduce NaOH requirements. The gradual reduction of pH from 6 to 5.5 resulted in 54.4 g/L succinic acid with 0.44 g/g yield and 0.82 g/L/h productivity and 43% lower NaOH consumption. The minimum selling price of succinic acid was estimated at $2.7/kg and $2.94/kg at annual production capacity of 50,000 t when the production cost of organic biowaste derived sugars were considered at $10/tTS and $110/tTS, respectively. The global warming potential and abiotic depletion potential were estimated at 2.72 kgCO2-eq/kgSA and 33.4 MJ/kgSA, respectively. Results showed that pH regulation is an important operating parameter towards the sustainable succinic acid production using municipal organic biowaste.

Impact of nitrogen deficiency on succinic acid production by engineered strains of Yarrowia lipolytica

Yarrowia lipolytica strains PGC01003 and PGC202 engineered for succinic acid production were studied and compared to the wild type strain W29. For the first time, these two strains were characterized in a chemically defined medium. Strain growth and organic acid production were investigated in fed-batch mode with glycerol as carbon and energy source. This study evaluated the impact of nitrogen deficiency strategy to redirect carbon flux toward succinic acid synthesis. Strain PGC01003 produced 19 g L−1 succinic acid with an overall yield of 0.23 g g−1 and an overall productivity of 0.23 g L−1 h−1, while strain PGC202 produced 33 g L−1 succinic acid with an overall yield of 0.12 g g−1 and a productivity of 0.57 g L−1 h−1. Nitrogen limitation effectively stopped biomass growth and increased succinic acid yield of PGC01003 and PGC202 by 18 % and 62 %, respectively. However, the specific succinic acid production rate was reduced by 77 % and 66 %, respectively.

Efficient biodegradation of aliphatic polyester by genetically engineered strains of the yeast Yarrowia lipolytica

The increased production and consumption of plastic materials in today's world has caused a huge number of environmental problems, the solution of which is now crucial to improving the state of nature of our planet. In this study, we expressed cutinases from Fusarium solani f. sp. pisi and from Trichoderma reesei, individually or in combination with overexpression of the native lipase Lip2 in the yeast Yarrowia lipolytica. The engineered strains showed biodegradation activity toward polyester in the pH range between 4.0 and 9.0. The highest esterase activity was observed at pH 9.0 for the strain overexpressing cutinase from F. solani and lipase from Y. lipolytica, it reached 63.5 U ml−1, moreover this strain was capable to degrade 0.5 g of polycaprolactone film within 144 h of shake flask culture. The obtained results showed that natural capability of Y. lipolytica combined with metabolic engineering results in a highly efficient biodegradation process.

A Yarrowia lipolytica Strain Engineered for Pyomelanin Production.

The yeast Yarrowia lipolytica naturally produces pyomelanin. This pigment accumulates in the extracellular environment following the autoxidation and polymerization of homogentisic acid, a metabolite derived from aromatic amino acids. In this study, we used a chassis strain optimized to produce aromatic amino acids for the de novo overproduction of pyomelanin. The gene 4HPPD, which encodes an enzyme involved in homogentisic acid synthesis (4-hydroxyphenylpyruvic acid dioxygenase), was characterized and overexpressed in the chassis strain with up to three copies, leading to pyomelanin yields of 4.5 g/L. Homogentisic acid is derived from tyrosine. When engineered strains were grown in a phenylalanine-supplemented medium, pyomelanin production increased, revealing that the yeast could convert phenylalanine to tyrosine, or that the homogentisic acid pathway is strongly induced by phenylalanine.

Probiotic and Immunomodulatory Activity of Marine Yeast Yarrowia lipolytica Strains and Response Against Vibrio parahaemolyticus in Fish.

Yarrowia lipolytica has been widely used in food industry but scarcely explored as probiotics. Thus, the aims of this study were to characterize in vitro the probiotic potential, antioxidant capacity, and antimicrobial activity of the marine yeast Y. lipolytica D-1 and N-6 strains. Dietary administration effect was evaluated in vivo on immunological parameters in serum, skin-mucus, intestine, and fish leukocytes upon challenge with Vibrio parahaemolyticus. The results showed that Y. lipolytica D-1 and N-6 strains grew with NaCl or bile salts but were sensitive to low pH. Each of the Y. lipolytica strains had a distinctive antioxidant capacity and fatty acid profile, but their antimicrobial activity was similar against fish bacterial pathogens. Fish (Lutjanus peru) supplemented with Y. lipolytica strains showed normal intestinal morphology, high IgM levels, and antioxidant enzyme activities. Immune-related genes were modulated in fish fed Y. lipolytica in a strain-dependent fashion. In addition, leucocytes from fish fed Y. lipolytica challenged with V. parahaemolyticus increased innate immune and antioxidant parameters compared with the control groups. In conclusion, the marine yeast Y. lipolytica D-1 and N-6 strains may be potential probiotics for fish by exerting free-radical scavenging, antimicrobial activity, and improved immune-protective responses against V. parahaemolyticus infection.

Adaptive laboratory evolution of Yarrowia lipolytica improves ferulic acid tolerance

Yarrowia lipolytica strain is a promising cell factory for the conversion of lignocellulose to biofuels and bioproducts. Despite the inherent robustness of this strain, further improvements to lignocellulose-derived inhibitors toxicity tolerance of Y. lipolytica are also required to achieve industrial application. Here, adaptive laboratory evolution was employed with increasing concentrations of ferulic acid. The adaptive laboratory evolution experiments led to evolve Y. lipolytica strain yl-XYL + *FA*4 with increased tolerance to ferulic acid as compared to the parental strain. Specifically, the evolved strain could tolerate 1.5 g/L ferulic acid, whereas 0.5 g/L ferulic acid could cause about 90% lethality of the parental strain. Transcriptome analysis of the evolved strain revealed several targets underlying toxicity tolerance enhancements. YALI0_E25201g, YALI0_F05984g, YALI0_B18854g, and YALI0_F16731g were among the highest upregulated genes, and the beneficial contributions of these genes were verified via reverse engineering. Recombinant strains with overexpressing each of these four genes obtained enhanced tolerance to ferulic acid as compared to the control strain. Fortunately, recombinant strains with overexpression of YALI0_E25201g, YALI0_B18854g, and YALI0_F16731g individually also obtained enhanced tolerance to vanillic acid. Overall, this work demonstrated a whole strain improvement cycle by "non-rational" metabolic engineering and presented new targets to modify Y. lipolytica for microbial lignocellulose valorization. KEY POINTS: • Adaptive evolution improved the ferulic acid tolerance of Yarrowia lipolytica • Transcriptome sequence was applied to analyze the ferulic acid tolerate strain • Three genes were demonstrated for both ferulic acid and vanillic acid tolerance.

Current Challenges and Opportunities in Non-native Chemical Production by Engineered Yeasts.

Yeasts are promising industrial hosts for sustainable production of fuels and chemicals. Apart from efficient bioethanol production, yeasts have recently demonstrated their potential for biodiesel production from renewable resources. The fuel-oriented product profiles of yeasts are now expanding to include non-native chemicals with the advances in synthetic biology. In this review, current challenges and opportunities in yeast engineering for sustainable production of non-native chemicals will be discussed, with a focus on the comparative evaluation of a bioethanol-producing Saccharomyces cerevisiae strain and a biodiesel-producing Yarrowia lipolytica strain. Synthetic pathways diverging from the distinctive cellular metabolism of these yeasts guide future directions for product-specific engineering strategies for the sustainable production of non-native chemicals on an industrial scale.

Improvement of glutathione production by a metabolically engineered Yarrowia lipolytica strain using a small-scale optimization approach.

In this study, we aimed to maximize glutathione (GSH) production by a metabolically engineered Yarrowia lipolytica strain using a small-scale optimization approach. A three levels four factorial Box-Behnken Design was used to assess the effect of pH, inulin extract, yeast extract and ammonium sulfate concentrations on cell growth and to generate a mathematical model which predict optimal conditions to maximize biomass production and thus GSH titer. The obtained results revealed that only yeast and inulin extract concentrations significantly affect biomass production. Based on the generated model, a medium composed of 10g/L of yeast extract and 10g/L of inulin extract from Jerusalem artichoke was used to conduct batch cultures in 2 L bioreactor. After 48h of culture, the biomass and the glutathione titer increased by 55% (5.8 gDCW/L) and 61% (1011.4mg/L), respectively, as compared to non-optimized conditions. From the obtained results, it could be observed that the model established from small scale culture (i.e. 2mL) is able to predict performance at larger scale (i.e. 2 L bioreactor, two orders of magnitude scale-up). Moreover, the results highlight the ability of the optimized process to ensure high titer of glutathione using a low-cost carbon source.

Physiological Characterization of a Novel Wild-Type Yarrowia lipolytica Strain Grown on Glycerol: Effects of Cultivation Conditions and Mode on Polyols and Citric Acid Production

A new yeast wild-type Yarrowia lipolytica isolate presented efficient growth on glycerol. During flask cultures, nitrogen limitation led to the secretion of sugar-alcohols as the major metabolites of the process (mannitol, arabitol and erythritol), whereas insignificant quantities of citrate were synthesized. Although in some instances high initial glycerol concentrations were employed (≈150 g/L), remarkable glycerol assimilation and polyol secretion was observed. Total polyols ≈ 52 g/L (conversion yield on glycerol consumed = 0.43 g/g) was recorded in the flask experiments. The sugar-alcohol production bioprocess was successfully simulated with the aid of a modified Velhlust–Aggelis model that fitted very well with the experimental data, while optimized parameter values seemed to be quite consistent. In bioreactor trials, a noticeable metabolic shift towards citric acid production was observed, while simultaneously insignificant polyol quantities were produced. In fed-batch bioreactor experiments, a total citric acid quantity ≈ 102 g/L was recorded—one of the highest in the literature for wild-type Y. lipolytica strains. This metabolic transition was due to higher oxygen saturation into the medium that occurred in the bioreactor experiments compared with the flasks. Cellular lipids produced in the bioreactor trial contained higher concentrations of unsaturated fatty acids compared with those produced in flasks.

De novo production of resveratrol from glycerol by engineering different metabolic pathways in Yarrowia lipolytica

Resveratrol is a polyphenol with multiple applications in pharma, cosmetics and food. The aim of this study was to construct Yarrowia lipolytica strains able to produce resveratrol. For this purpose, resveratrol-biosynthesis genes from bacteria and plants were expressed in this host. Since resveratrol can be produced either via tyrosine or phenylaniline, both pathways were tested, first with a single copy and then with two copies. The phenylalanine pathway resulted in slightly higher production in glucose media, although when the media was supplemented with amino acids, the best production was found in the strain with two copies of the tyrosine pathway, which reached 0.085 ​g/L. When glucose was replaced by glycerol, a preferred substrate for bioproduction, the best results, 0.104 ​g/L, were obtained in a strain combining the expression of the two synthesis pathways. Finally, the best producer strain was tested in bioreactor conditions where a production of 0.43 ​g/L was reached. This study suggests that Y. lipolytica is a promising host for resveratrol production from glycerol.

Yarrowia lipolytica Strains Engineered for the Production of Terpenoids.

Terpenoids are a diverse group of over 55,000 compounds with potential applications as advanced fuels, bulk and fine chemicals, pharmaceutical ingredients, agricultural chemicals, etc. To facilitate their bio-based production, there is a need for plug-and-play hosts, capable of high-level production of different terpenoids. Here we engineer Yarrowia lipolytica platform strains for the overproduction of mono-, sesqui-, di-, tri-, and tetraterpenoids. The monoterpene platform strain was evaluated by expressing Perilla frutescens limonene synthase, which resulted in limonene titer of 35.9 mg/L and was 100-fold higher than when the same enzyme was expressed in the strain without mevalonate pathway improvement. Expression of Callitropsis nootkatensis valencene synthase in the sesquiterpene platform strain resulted in 113.9 mg/L valencene, an 8.4-fold increase over the control strain. Platform strains for production of squalene, complex triterpenes, or diterpenes and carotenoids were also constructed and resulted in the production of 402.4 mg/L squalene, 22 mg/L 2,3-oxidosqualene, or 164 mg/L β-carotene, respectively. The presented terpenoid platform strains can facilitate the evaluation of terpenoid biosynthetic pathways and are a convenient starting point for constructing efficient cell factories for the production of various terpenoids. The platform strains and exemplary terpenoid strains can be obtained from Euroscarf.

Alpha-Ketoglutaric Acid Production from a Mixture of Glycerol and Rapeseed Oil by Yarrowia lipolytica Using Different Substrate Feeding Strategies

The microbiological biosynthesis of α-ketoglutaric acid (KGA) has recently captured the attention of many scientists as an alternative to its common chemical synthesis. The present study aimed to evaluate the effect of the feeding strategy of substrates, i.e., glycerol (G = 20 g·dm−3) and rapeseed oil (O = 20 g·dm−3), on yeast growth and the parameters of KGA biosynthesis by a wild strain Yarrowia lipolytica A-8 in fed-batch and repeated-batch cultures. The effectiveness of KGA biosynthesis was demonstrated to depend on thiamine concentration and the substrate feeding method. In the fed-batch culture incubated with 3 µg·dm−3 of thiamine and a substrate feeding variant 2G(_OGO), KGA was produced in the amount of 62.1 g·dm−3 at the volumetric production rate of 0.37 g·dm−3·h−1. These values of KGA production parameters were higher than these obtained in the control culture (with rapeseed oil only). During 10 cycles of the 1788-h repeated-batch culture carried out acc. to the feeding strategy 2G(_OGO), in the last 5 cycles the yeast produced from 55.6 to 58.2 g·dm−3 of KGA and maximally 2.9 g·dm−3 of the pyruvic acid as a by-product.