Phospholipids (PLs) are a class of lipids with many proven biological functions. They are commonl... more Phospholipids (PLs) are a class of lipids with many proven biological functions. They are commonly used in lipid replacement therapy to enrich cell membranes damaged in chronic neurodegenerative diseases, cancer, or aging processes. Due to their amphipathic nature, PLs have been widely used in food, cosmetic, and pharmaceutical products as natural emulsifiers and components of liposomes. In Yarrowia lipolytica, PLs are synthesized through a similar pathway like in higher eukaryotes. However, PL biosynthesis in this yeast is still poorly understood. The key intermediate in this pathway is phosphatidic acid, which in Y. lipolytica is mostly directed to the production of triacylglycerols and, in a lower amount, to PL. This study aimed to deliver a strain with improved PL production, with a particular emphasis on increased biosynthesis of phosphatidylcholine (PC). Several genetic modifications were performed: overexpression of genes from PL biosynthesis pathways as well as the deletion ...
Yarrowia lipolytica yeast are able to produce kynurenic acid—a very valuable compound acting as a... more Yarrowia lipolytica yeast are able to produce kynurenic acid—a very valuable compound acting as a neuroprotective and antioxidant agent in humans. The recent data proved the existence of the kynurenine biosynthesis pathway in this yeast cells. Due to this fact, the aim of this work was to enhance kynurenic acid production using crude glycerol and soybean molasses as cheap and renewable carbon and nitrogen sources. The obtained results showed that Y. lipolytica GUT1 mutants are able to produce kynurenic acid in higher concentrations (from 4.5 mg dm−3 to 14.1 mg dm−3) than the parental strain (3.6 mg dm−3) in the supernatant in a medium with crude glycerol. Moreover, the addition of soybean molasses increased kynurenic acid production by using wild type and transformant strains. The A-101.1.31 GUT1/1 mutant strain produced 17.7 mg dm−3 of kynurenic acid in the supernatant during 150 h of the process and 576.7 mg kg−1 of kynurenic acid in dry yeast biomass. The presented work proves th...
Invertases are important enzymes used in the food industry. Despite many studies on the invertase... more Invertases are important enzymes used in the food industry. Despite many studies on the invertase-encoding SUC2 gene expression in the industrial yeast Yarrowia lipolytica, no biochemical characteristics of this enzyme expressed as heterologous protein have been provided. Here, two isoforms of extracellular invertase produced by Y. lipolytica were detected using ion-exchange chromatography. Specific activities of 226.45 and 432.66 U/mg for the first and second isoform, respectively, were determined. Basic characteristics of this enzyme were similar to the one isolated from Saccharomyces cerevisiae (optimum pH and temperature, metal ions inhibition, substrate specificity and fructooligosaccharides (FOS) biosynthesis). The apparent differences were higher KM for sucrose (67 mM) and lower molecular mass (66 kDa) resulting from lower N-glycosylation level (9.1% of mass). The N-glycan structures determined by MALDI-TOF and HPLC represented high mannose structures, though with much shorte...
The present study aimed to develop a technology for the production of dietary supplements based o... more The present study aimed to develop a technology for the production of dietary supplements based on yeast biomass and α-ketoglutaric acid (KGA), produced by a new transformant of Yarrowia lipolytica with improved KGA biosynthesis ability, as well to verify the usefulness of the obtained products for food and feed purposes. Transformants of Y. lipolytica were constructed to overexpress genes encoding glycerol kinase, methylcitrate synthase and mitochondrial organic acid transporter. The strains were compared in terms of growth ability in glycerol- and oil-based media as well as their suitability for KGA biosynthesis in mixed glycerol–oil medium. The impact of different C:N:P ratios on KGA production by selected strain was also evaluated. Application of the strain that overexpressed all three genes in the culture with a C:N:P ratio of 87:5:1 allowed us to obtain 53.1 g/L of KGA with productivity of 0.35 g/Lh and yield of 0.53 g/g. Finally, the possibility of obtaining three different p...
Additional file 2: Table S2. Amino acids composition of the yeast biomass of Yarrowia clade speci... more Additional file 2: Table S2. Amino acids composition of the yeast biomass of Yarrowia clade species growing on crude glycerol from biodiesel industry in bioreactor cultures.
Additional file 5: Construction of the Y. lipolytica strains in which the scGAL genes were overex... more Additional file 5: Construction of the Y. lipolytica strains in which the scGAL genes were overexpressed. The auxotrophic PO1d strain was used as the acceptor strain. The genes were inserted one by one to create strains that overexpressed different combination of the scGAL genes; URA3ex and LEU2ex were used as selection markers. To recover auxotrophies necessary to continuing the transformation process, a JME547 plasmid containing Cre-Lox recombinase was used to transform Y. lipolytica Y3683, thus generating strain Y3686 [35]. In turn, to recover prototrophy in the Y. lipolytica Y3687 strain, a purified SalI fragment of the pINA62 plasmid that contained the LEU2 gene was introduced [28].
Additional file 2: Overexpression of scGAL and ylGAL genes in Y. lipolytica. Growth of Y. lipolyt... more Additional file 2: Overexpression of scGAL and ylGAL genes in Y. lipolytica. Growth of Y. lipolytica transformants containing scGAL genes (scGAL1,7,10) on YNB medium containing 1 % galactose (A); yeast were incubated for 1 week at 28 °C. Growth of Y. lipolytica transformants overexpressing different combinations of ylGAL genes on YNB medium containing 1 % galactose (B); yeast were incubated for 2 weeks at 28 °C.
Additional file 3: Sugar consumption by S. cerevisiae null mutants expressing Y. lipolytica hexos... more Additional file 3: Sugar consumption by S. cerevisiae null mutants expressing Y. lipolytica hexose transporters after 72 h of growth in YNB medium containing 1 % glucose (■) or 1 % galactose (■) (A). Expression profiles of Y. lipolytica genes encoding hexose transporters in the W29 and Y4588 strains (B). Yeast were incubated for 3 h in YNB medium containing 1.0 % glucose or 1.0 % galactose. The amplification of the PCR fragment in the genomic DNA served as a control for the primers' efficiency. Abbreviations: In - inoculum.
In this study, an attempt was made to develop shortcrust pastries containing different amounts of... more In this study, an attempt was made to develop shortcrust pastries containing different amounts of chokeberry pomace (0%, 10%, 30%, 50%), modulating their degree of sweetness via the application of sucrose or erythritol. The obtained products were assessed for their nutritional value (energy value, protein, fats, dietary fibre, sugars, minerals). Bioactive compounds, as well as antioxidant and anti-diabetic properties in an in vitro model and sensory attributes, were also analysed. Increasing the proportion of chokeberry pomace in shortcrust pastries improved their nutritional value, especially their energy value (reduction of nearly 30% for shortcrust pastries with 50% pomace sweetened with erythritol), nutritional fibre content (10-fold higher in shortcrust pastries with the highest proportion of pomace) and potassium, calcium, magnesium, and iron content. Chokeberry pomace was also a carrier of 14 bioactive compounds. The most beneficial antioxidant and anti-diabetic effect was sh...
Most biosurfactants are obtained using costly culture media and purification processes, which lim... more Most biosurfactants are obtained using costly culture media and purification processes, which limits their wider industrial use. Sustainability of their production processes can be achieved, in part, by using cheap substrates found among agricultural and food wastes or byproducts. In the present study, crude glycerol, a raw material obtained from several industrial processes, was evaluated as a potential low-cost carbon source to reduce the costs of surfactin production by Bacillus subtilis #309. The culture medium containing soap-derived waste glycerol led to the best surfactin production, reaching about 2.8 g/L. To the best of our knowledge, this is the first report describing surfactin production by B. subtilis using stearin and soap wastes as carbon sources. A complete chemical characterization of surfactin analogs produced from the different waste glycerol samples was performed by liquid chromatography–mass spectrometry (LC-MS) and Fourier transform infrared spectroscopy (FTIR)...
Abstract The global market for high-value fatty acids production, mainly omega-3/6, hydroxy fatty... more Abstract The global market for high-value fatty acids production, mainly omega-3/6, hydroxy fatty-acids, waxes and their derivatives, has seen strong development in the last decade. The reason for this growth was the increasing utilization of these lipids as significant ingredients for cosmetics, food and the oleochemical industries. The large demand for these compounds resulted in a greater scientific interest in research focused on alternative sources of oil production - among which microorganisms attracted the most attention. Microbial oil production offers the possibility to engineer the pathways and store lipids enriched with the desired fatty acids. Moreover, costly chemical steps are avoided and direct commercial use of these fatty acids is available. Among all microorganisms, the oleaginous yeasts have become the most promising hosts for lipid production - their efficient lipogenesis, ability to use various (often highly affordable) carbon sources, feasible large-scale cultivations and wide range of available genetic engineering tools turns them into powerful micro-factories. This review is an in-depth description of the recent developments in the engineering of the lipid biosynthetic pathway with oleaginous yeasts. The different classes of valuable lipid compounds with their derivatives are described and their importance for human health and industry is presented. The emphasis is also placed on the optimization of culture conditions in order to improve the yield and titer of these valuable compounds. Furthermore, the important economic aspects of the current microbial oil production are discussed.
Yarrowia lipolytica is a non-conventional yeast producing valuable compounds, such as citric acid... more Yarrowia lipolytica is a non-conventional yeast producing valuable compounds, such as citric acids, from renewable raw materials. This study investigated the impact of citrate synthase overexpression on the biosynthesis of citric and isocitric acid in Y. lipolytica. Two transformants of Y. lipolytica A101.1.31 strain (efficient citric acid producer), overexpressing CIT1 or CIT2 gene (encoding proteins with citrate synthase activity), were constructed. The results revealed that overexpression of either of these genes enhances citrate synthase activity. Additionally, the cit1 knockout strain was unable to use propionate as the sole carbon source, which proves that CIT1 gene encodes a dual activity protein–citrate and 2-methylcitrate synthase. In the overexpressing mutants, a significant increase in isocitric acid biosynthesis was observed. Both CIT1 and CIT2 overexpressing strains produced citric and isocitric acid from vegetable oil in a ratio close to 1 (CA/ICA ratio for wild-type s...
Phospholipids (PLs) are a class of lipids with many proven biological functions. They are commonl... more Phospholipids (PLs) are a class of lipids with many proven biological functions. They are commonly used in lipid replacement therapy to enrich cell membranes damaged in chronic neurodegenerative diseases, cancer, or aging processes. Due to their amphipathic nature, PLs have been widely used in food, cosmetic, and pharmaceutical products as natural emulsifiers and components of liposomes. In Yarrowia lipolytica, PLs are synthesized through a similar pathway like in higher eukaryotes. However, PL biosynthesis in this yeast is still poorly understood. The key intermediate in this pathway is phosphatidic acid, which in Y. lipolytica is mostly directed to the production of triacylglycerols and, in a lower amount, to PL. This study aimed to deliver a strain with improved PL production, with a particular emphasis on increased biosynthesis of phosphatidylcholine (PC). Several genetic modifications were performed: overexpression of genes from PL biosynthesis pathways as well as the deletion ...
Yarrowia lipolytica yeast are able to produce kynurenic acid—a very valuable compound acting as a... more Yarrowia lipolytica yeast are able to produce kynurenic acid—a very valuable compound acting as a neuroprotective and antioxidant agent in humans. The recent data proved the existence of the kynurenine biosynthesis pathway in this yeast cells. Due to this fact, the aim of this work was to enhance kynurenic acid production using crude glycerol and soybean molasses as cheap and renewable carbon and nitrogen sources. The obtained results showed that Y. lipolytica GUT1 mutants are able to produce kynurenic acid in higher concentrations (from 4.5 mg dm−3 to 14.1 mg dm−3) than the parental strain (3.6 mg dm−3) in the supernatant in a medium with crude glycerol. Moreover, the addition of soybean molasses increased kynurenic acid production by using wild type and transformant strains. The A-101.1.31 GUT1/1 mutant strain produced 17.7 mg dm−3 of kynurenic acid in the supernatant during 150 h of the process and 576.7 mg kg−1 of kynurenic acid in dry yeast biomass. The presented work proves th...
Invertases are important enzymes used in the food industry. Despite many studies on the invertase... more Invertases are important enzymes used in the food industry. Despite many studies on the invertase-encoding SUC2 gene expression in the industrial yeast Yarrowia lipolytica, no biochemical characteristics of this enzyme expressed as heterologous protein have been provided. Here, two isoforms of extracellular invertase produced by Y. lipolytica were detected using ion-exchange chromatography. Specific activities of 226.45 and 432.66 U/mg for the first and second isoform, respectively, were determined. Basic characteristics of this enzyme were similar to the one isolated from Saccharomyces cerevisiae (optimum pH and temperature, metal ions inhibition, substrate specificity and fructooligosaccharides (FOS) biosynthesis). The apparent differences were higher KM for sucrose (67 mM) and lower molecular mass (66 kDa) resulting from lower N-glycosylation level (9.1% of mass). The N-glycan structures determined by MALDI-TOF and HPLC represented high mannose structures, though with much shorte...
The present study aimed to develop a technology for the production of dietary supplements based o... more The present study aimed to develop a technology for the production of dietary supplements based on yeast biomass and α-ketoglutaric acid (KGA), produced by a new transformant of Yarrowia lipolytica with improved KGA biosynthesis ability, as well to verify the usefulness of the obtained products for food and feed purposes. Transformants of Y. lipolytica were constructed to overexpress genes encoding glycerol kinase, methylcitrate synthase and mitochondrial organic acid transporter. The strains were compared in terms of growth ability in glycerol- and oil-based media as well as their suitability for KGA biosynthesis in mixed glycerol–oil medium. The impact of different C:N:P ratios on KGA production by selected strain was also evaluated. Application of the strain that overexpressed all three genes in the culture with a C:N:P ratio of 87:5:1 allowed us to obtain 53.1 g/L of KGA with productivity of 0.35 g/Lh and yield of 0.53 g/g. Finally, the possibility of obtaining three different p...
Additional file 2: Table S2. Amino acids composition of the yeast biomass of Yarrowia clade speci... more Additional file 2: Table S2. Amino acids composition of the yeast biomass of Yarrowia clade species growing on crude glycerol from biodiesel industry in bioreactor cultures.
Additional file 5: Construction of the Y. lipolytica strains in which the scGAL genes were overex... more Additional file 5: Construction of the Y. lipolytica strains in which the scGAL genes were overexpressed. The auxotrophic PO1d strain was used as the acceptor strain. The genes were inserted one by one to create strains that overexpressed different combination of the scGAL genes; URA3ex and LEU2ex were used as selection markers. To recover auxotrophies necessary to continuing the transformation process, a JME547 plasmid containing Cre-Lox recombinase was used to transform Y. lipolytica Y3683, thus generating strain Y3686 [35]. In turn, to recover prototrophy in the Y. lipolytica Y3687 strain, a purified SalI fragment of the pINA62 plasmid that contained the LEU2 gene was introduced [28].
Additional file 2: Overexpression of scGAL and ylGAL genes in Y. lipolytica. Growth of Y. lipolyt... more Additional file 2: Overexpression of scGAL and ylGAL genes in Y. lipolytica. Growth of Y. lipolytica transformants containing scGAL genes (scGAL1,7,10) on YNB medium containing 1 % galactose (A); yeast were incubated for 1 week at 28 °C. Growth of Y. lipolytica transformants overexpressing different combinations of ylGAL genes on YNB medium containing 1 % galactose (B); yeast were incubated for 2 weeks at 28 °C.
Additional file 3: Sugar consumption by S. cerevisiae null mutants expressing Y. lipolytica hexos... more Additional file 3: Sugar consumption by S. cerevisiae null mutants expressing Y. lipolytica hexose transporters after 72 h of growth in YNB medium containing 1 % glucose (■) or 1 % galactose (■) (A). Expression profiles of Y. lipolytica genes encoding hexose transporters in the W29 and Y4588 strains (B). Yeast were incubated for 3 h in YNB medium containing 1.0 % glucose or 1.0 % galactose. The amplification of the PCR fragment in the genomic DNA served as a control for the primers' efficiency. Abbreviations: In - inoculum.
In this study, an attempt was made to develop shortcrust pastries containing different amounts of... more In this study, an attempt was made to develop shortcrust pastries containing different amounts of chokeberry pomace (0%, 10%, 30%, 50%), modulating their degree of sweetness via the application of sucrose or erythritol. The obtained products were assessed for their nutritional value (energy value, protein, fats, dietary fibre, sugars, minerals). Bioactive compounds, as well as antioxidant and anti-diabetic properties in an in vitro model and sensory attributes, were also analysed. Increasing the proportion of chokeberry pomace in shortcrust pastries improved their nutritional value, especially their energy value (reduction of nearly 30% for shortcrust pastries with 50% pomace sweetened with erythritol), nutritional fibre content (10-fold higher in shortcrust pastries with the highest proportion of pomace) and potassium, calcium, magnesium, and iron content. Chokeberry pomace was also a carrier of 14 bioactive compounds. The most beneficial antioxidant and anti-diabetic effect was sh...
Most biosurfactants are obtained using costly culture media and purification processes, which lim... more Most biosurfactants are obtained using costly culture media and purification processes, which limits their wider industrial use. Sustainability of their production processes can be achieved, in part, by using cheap substrates found among agricultural and food wastes or byproducts. In the present study, crude glycerol, a raw material obtained from several industrial processes, was evaluated as a potential low-cost carbon source to reduce the costs of surfactin production by Bacillus subtilis #309. The culture medium containing soap-derived waste glycerol led to the best surfactin production, reaching about 2.8 g/L. To the best of our knowledge, this is the first report describing surfactin production by B. subtilis using stearin and soap wastes as carbon sources. A complete chemical characterization of surfactin analogs produced from the different waste glycerol samples was performed by liquid chromatography–mass spectrometry (LC-MS) and Fourier transform infrared spectroscopy (FTIR)...
Abstract The global market for high-value fatty acids production, mainly omega-3/6, hydroxy fatty... more Abstract The global market for high-value fatty acids production, mainly omega-3/6, hydroxy fatty-acids, waxes and their derivatives, has seen strong development in the last decade. The reason for this growth was the increasing utilization of these lipids as significant ingredients for cosmetics, food and the oleochemical industries. The large demand for these compounds resulted in a greater scientific interest in research focused on alternative sources of oil production - among which microorganisms attracted the most attention. Microbial oil production offers the possibility to engineer the pathways and store lipids enriched with the desired fatty acids. Moreover, costly chemical steps are avoided and direct commercial use of these fatty acids is available. Among all microorganisms, the oleaginous yeasts have become the most promising hosts for lipid production - their efficient lipogenesis, ability to use various (often highly affordable) carbon sources, feasible large-scale cultivations and wide range of available genetic engineering tools turns them into powerful micro-factories. This review is an in-depth description of the recent developments in the engineering of the lipid biosynthetic pathway with oleaginous yeasts. The different classes of valuable lipid compounds with their derivatives are described and their importance for human health and industry is presented. The emphasis is also placed on the optimization of culture conditions in order to improve the yield and titer of these valuable compounds. Furthermore, the important economic aspects of the current microbial oil production are discussed.
Yarrowia lipolytica is a non-conventional yeast producing valuable compounds, such as citric acid... more Yarrowia lipolytica is a non-conventional yeast producing valuable compounds, such as citric acids, from renewable raw materials. This study investigated the impact of citrate synthase overexpression on the biosynthesis of citric and isocitric acid in Y. lipolytica. Two transformants of Y. lipolytica A101.1.31 strain (efficient citric acid producer), overexpressing CIT1 or CIT2 gene (encoding proteins with citrate synthase activity), were constructed. The results revealed that overexpression of either of these genes enhances citrate synthase activity. Additionally, the cit1 knockout strain was unable to use propionate as the sole carbon source, which proves that CIT1 gene encodes a dual activity protein–citrate and 2-methylcitrate synthase. In the overexpressing mutants, a significant increase in isocitric acid biosynthesis was observed. Both CIT1 and CIT2 overexpressing strains produced citric and isocitric acid from vegetable oil in a ratio close to 1 (CA/ICA ratio for wild-type s...
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