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.

Biosynthesis of Fatty Acid Derivatives by Recombinant Yarrowia lipolytica Containing MsexD2 and MsexD3 Desaturase Genes from Manduca sexta.

One of the most interesting groups of fatty acid derivates is the group of conjugated fatty acids from which the most researched include: conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), which are associated with countless health benefits. Sex pheromone mixtures of some insect species, including tobacco horn-worm (Manduca sexta), are typical for the production of uncommon C16 long conjugated fatty acids with two and three conjugated double bonds, as opposed to C18 long CLA and CLNA. In this study, M. sexta desaturases MsexD2 and MsexD3 were expressed in multiple strains of Y. lipolytica with different genotypes. Experiments with the supplementation of fatty acid methyl esters into the medium resulted in the production of novel fatty acids. Using GCxGC-MS, 20 new fatty acids with two or three double bonds were identified. Fatty acids with conjugated or isolated double bonds, or a combination of both, were produced in trace amounts. The results of this study prove that Y. lipolytica is capable of synthesizing C16-conjugated fatty acids. Further genetic optimization of the Y. lipolytica genome and optimization of the fermentation process could lead to increased production of novel fatty acid derivatives with biotechnologically interesting properties.

Correlation between α-synuclein and fatty acid composition in jejunum of rotenone-treated mice is dependent on acyl chain length.

Events associated with the progression of Parkinson´s disease (PD) are closely related to biomembrane dysfunction. The specific role of membrane composition in the conformational stability of alpha synuclein (αS) has already been well documented. Administration of rotenone is one of the best strategies to initiate PD phenotype in animal models. In the present study, daily exposure (14 weeks) of orally administered rotenone (10 mg/kg) was employed in a mouse model. The mitochondrial complex I inhibition resulted in elevated level of αS in whole tissue homogenate of mouse jejunum. In addition, we identified a strong intra-individual correlation between αS level and the specific esterified fatty acids. The observed correlation depends mainly on the acyl chain length. Based on the obtained results, it is suggested that there is a high potential to manipulate fatty acid homeostasis in modulating αS based pathogenesis of PD, at least in experimental conditions.

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.

Impairment of carotenoid biosynthesis through CAR1 gene mutation results in CoQ10, sterols, and phytoene accumulation in Rhodotorula mucilaginosa.

Red yeasts, mainly included in the genera Rhodotorula, Rhodosporidiobolus, and Sporobolomyces, are renowned biocatalysts for the production of a wide range of secondary metabolites of commercial interest, among which lipids, carotenoids, and other isoprenoids. The production of all these compounds is tightly interrelated as they share acetyl-CoA and the mevalonate pathway as common intermediates. Here, T-DNA insertional mutagenesis was applied to the wild type strain C2.5t1 of Rhodotorula mucilaginosa for the isolation of albino mutants with impaired carotenoids biosynthesis. The rationale behind this approach was that a blockage in carotenoid biosynthetic pathway could divert carbon flux toward the production of lipids and/or other molecules deriving from terpenoid precursors. One characterized albino mutant, namely, strain W4, carries a T-DNA insertion in the CAR1 gene coding for phytoene desaturase. When cultured in glycerol-containing medium, W4 strain showed significant decreases in cell density and fatty acids content in respect to the wild type strain. Conversely, it reached significantly higher productions of phytoene, CoQ10, and sterols. These were supported by an increased expression of CAR2 gene that codes for phytoene synthase/lycopene cyclase. Thus, in accordance with the starting hypothesis, the impairment of carotenoids biosynthesis can be explored to pursue the biotechnological exploitation of red yeasts for enhanced production of secondary metabolites with several commercial applications. KEY POINTS: • The production of lipids, carotenoids, and other isoprenoids is tightly interrelated. • CAR1 gene mutation results in the overproduction of phytoene, CoQ10, and sterols. • Albino mutants are promising tools for the production of secondary metabolites.

Animal Fat as a Substrate for Production of n-6 Fatty Acids by Fungal Solid-State Fermentation.

The method of solid-state fermentation (SSF) represents a powerful technology for the fortification of animal-based by-products. Oleaginous Zygomycetes fungi are efficient microbial cell factories used in SSF to valorize a wide range of waste and rest cereal materials. The application of this fermentation technique for utilization and biotransformation of animal-based materials represents a distinguished step in their treatment. In this study, for the first time, the strain Umbelopsis isabellina CCF2412 was used for the bioconversion of animal fat by-products to the fermented bioproducts enriched with n-6 polyunsaturated fatty acids, mainly γ-linolenic acid (GLA). Bioconversion of both cereals and the animal fat by-product resulted in the production of fermented bioproducts enriched with not just GLA (maximal yield was 6.4 mg GLA/g of fermented bioproduct), but also with high yields of glucosamine. Moreover, the fermentation on the cornmeal matrix led to obtaining bioproduct enriched with ß-carotene. An increased amount of ß-carotene content improved the antioxidant stability of obtained fermented bioproducts. Furthermore, the application of Fourier-transform infrared spectroscopy for rapid analysis and characterization of the biochemical profile of obtained SSF bioproducts was also studied.

Fatty acid composition in seeds of holoparasitic Orobanchaceae from the Caucasus region: Relation to species, climatic conditions and nutritional value.

The specialization of parasitic plants from the Orobanchaceae family to the heterotrophic lifestyle caused several morphological, physiological and molecular changes. One of the adaptations to the parasitic lifestyle is the production of a large number of the smallest seeds in world flora, also called "dust-seeds". Seeds of 34 holoparasitic species from the Cistanche, Orobanche, Phelipanche, and Phelypaea genera were collected in the Caucasus region (54 samples) and their fatty acid content and compositions analysed. Of these seeds, 28 were investigated for the first time, and 12 are endemic to the Caucasus (one of the most important biodiversity hotspots in the world). The influence of different hosts, populations, habitats, and climatic conditions on the fatty acid content and composition, as well as some connections of taxonomic classification are discussed. The fatty acid content in the species varied between 0.9 and 42.5%, and showed quantitative differences at generic and infrageneric levels, while displaying uniform fatty acid composition. Thirteen fatty acids were identified, of which nine were undescribed for Orobanchaceae. The fatty acid composition of the Orobanchaceae seeds represented a mixture of saturated fatty acids (SFAs) (average 7.8%) and unsaturated fatty acids (UFAs) (average 92.2%). The fatty acid content in the Orobanchaceae seeds was directly unrelated to taxonomy, while the n-6/n-3 fatty acid ratio supported the clear separation of the Orobanche and Phelipanche genera. Orobanchaceae seeds contained mainly linoleic and oleic acids, thus they could be a potential nutritional source of the unsaturated fatty acids. Additionally, the studies confirmed the hypothesis that the degree of seed oil fatty acid unsaturation increased in colder climatic conditions, especially for the Orobanche genus.

Biotransformation of Animal Fat-By Products into ARA-Enriched Fermented Bioproducts by Solid-State Fermentation of Mortierella alpina.

Solid-state fermentation (SSF) is a powerful fermentation technology for valorizing rest materials and by-products of different origin. Oleaginous Zygomycetes fungi are often used in SSF as an effective cell factory able to valorize a wide range of hydrophilic and hydrophobic substrates and produce lipid-enriched bioproducts. In this study, for the first time, the strain Mortierella alpina was used in SSF for the bioconversion of animal fat by-products into high value fermented bioproducts enriched with arachidonic acid (ARA). Two cereals-based matrixes mixed with four different concentrations of animal fat by-product were evaluated for finding optimal conditions of a fat-based SSF. All obtained fermented bioproducts were found to be enriched with ARA. The highest substrate utilization (25.8%) was reached for cornmeal and it was almost double than for the respective wheat bran samples. Similarly, total fatty acid content in a fermented bioproduct prepared on cornmeal is almost four times higher in contrast to wheat bran-based bioproduct. Although in general the addition of an animal fat by-product caused a gradual cessation of ARA yield in the obtained fermented bioproduct, the content of ARA in fungal biomass was higher. Thus, M. alpina CCF2861 effectively transformed exogenous fatty acids from animal fat substrate to ARA. Maximum yield of 32.1 mg of ARA/g of bioproduct was reached when using cornmeal mixed with 5% (w/w) of an animal fat by-product as substrate. Furthermore, implementation of attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy in characterization of obtained SSF bioproducts was successfully tested as an alternative tool for complex analysis, compared to traditional time-consuming methods.

Effect of Solid-State Fermented Wheat Bran Supplemented with Agrimony Extract on Growth Performance, Fatty Acid Profile, and Meat Quality of Broiler Chickens.

The impact of the broiler diet modification on the following parameters was evaluated: meat quality, carcass traits, and bone and blood parameters. One hundred twenty one-day-old COBB 500 broiler chickens were assigned to three experimental groups (40 birds per group) with four replications (10 per pen) for 35 days of fattening. The control (C) was fed a basic feed mixture. The diet supplemented with 10% of fermented feed (FF10) and additionally supported by 0.2% of agrimony extract (FF10 + AE) was applied to the second and third groups, respectively. FF10 showed both a lower average daily feed intake and total feed consumption when compared to that of C (p < 0.05). Lower concentration of alkaline-phosphatase and calcium and higher total lipids and triglycerides in blood were observed in FF10 + AE. Breast and thigh meat showed a lower content of polyunsaturated fatty acid n-3 and n-6 in the FF10 + AE group (p < 0.01). The increase of gamma-linolenic acid content in breast and thigh meat samples obtained from the experimental groups was significant (p < 0.001 and p < 0.05; respectively). Lower lipid oxidation was observed in the thigh muscle of the FF10 + AE group on the first day of storage (p < 0.01). The current study indicates that FF10 + AE supplementation can be successfully applied to enhance broiler performance and meat quality.

Microcultivation and FTIR spectroscopy-based screening revealed a nutrient-induced co-production of high-value metabolites in oleaginous Mucoromycota fungi.

Mucoromycota fungi possess a versatile metabolism and can utilize various substrates for production of industrially important products, such as lipids, chitin/chitosan, polyphosphates, pigments, alcohols and organic acids. However, as far as commercialisation is concerned, establishing industrial biotechnological processes based on Mucoromycota fungi is still challenging due to the high production costs compared to the final product value. Therefore, the development of co-production concept is highly desired since more than one valuable product could be produced at the time and the process has a potentially higher viability. To develop such biotechnological strategy, we applied a high throughput approach consisting of micro-titre cultivation and FTIR spectroscopy. This approach allows single-step biochemical fingerprinting of either fungal biomass or growth media without tedious extraction of metabolites. The influence of two types of nitrogen sources and different levels of inorganic phosphorus on the co-production of lipids, chitin/chitosan and polyphosphates for nine different oleaginous Mucoromycota fungi was evaluated. FTIR analysis of biochemical composition of Mucoromycota fungi and biomass yield showed that variation in inorganic phosphorus had higher effect when inorganic nitrogen source-ammonium sulphate-was used. It was observed that: (1) Umbelopsis vinacea reached almost double biomass yield compared to other strains when yeast extract was used as nitrogen source while phosphorus limitation had little effect on the biomass yield; (2) Mucor circinelloides, Rhizopus stolonifer, Amylomyces rouxii, Absidia glauca and Lichtheimia corymbifera overproduced chitin/chitosan under the low pH caused by the limitation of inorganic phosphorus; (3) Mucor circinelloides, Amylomyces rouxii, Rhizopus stolonifer and Absidia glauca were able to store polyphosphates in addition to lipids when high concentration of inorganic phosphorus was used; (4) the biomass and lipid yield of high-value lipid producers Mortierella alpina and Mortierella hyalina were significantly increased when high concentrations of inorganic phosphorus were combined with ammonium sulphate, while the same amount of inorganic phosphorus combined with yeast extract showed negative impact on the growth and lipid accumulation. FTIR spectroscopy revealed the co-production potential of several oleaginous Mucoromycota fungi forming lipids, chitin/chitosan and polyphosphates in a single cultivation process.

Conversion of waste materials into very long chain fatty acids by the recombinant yeast Yarrowia lipolytica.

Erucic acid (C22:1Δ13) has several industrial applications including its use as a lubricant, surfactant and biodiesel and composite material constituent. It is produced by plants belonging to the Brassicaceae family, especially by the high erucic acid rapeseed. The ability to convert oleic acid into erucic acid is facilitated by FAE1. In this study, FAD2 (encoding Δ12-desaturase) was deleted in the strain Po1d to increase oleic acid content. Subsequently, FAE1 from Thlaspi arvense was overexpressed in Yarrowia lipolytica with the Δfad2 genotype. This resulted in the YL10 strain producing very long chain fatty acids, especially erucic acid. The YL10 strain was cultivated in media containing crude glycerol and waste cooking oil as carbon substrates. The cells grown using glycerol produced microbial oil devoid of linoleic acid, which was enriched with very long chain fatty acids, mainly erucic acid (9% of the total fatty acids). When cells were grown using waste cooking oil, the highest yield of erucic acid was obtained (887 mg L-1). However, external linoleic and α-linolenic were accumulated in cellular lipids when yeasts were grown in an oil medium. This study describes the possibility of conversion of waste material into erucic acid by a recombinant yeast strain.

Dual production of polyunsaturated fatty acids and beta-carotene with Mucor wosnessenskii by the process of solid-state fermentation using agro-industrial waste.

Solid-state fermentation is a technique employing microorganisms grown on a solid substrate in the absence of free water. The substrates used in this process are mostly waste from the agro-industry (brans, spent malt grains, distiller grains, etc.) that improves not only the economy of the process but also has positive effect on waste management problems. Zygomycetous fungi are not only able to grow in such conditions but also enrich fermented materials with various types of bioactive compounds. Mucor sp. strains have been identified as producers of gamma-linolenic acid and beta-carotene in submerged fermentation. The aim of the present study was to identify the best microbial producer of gamma-linolenic acid and beta-carotene among four different Mucor strains and to study the requirements for the dual production of these metabolites. Mucor wosnessenskii was identified as the most suitable producer of both metabolites. After optimization of the fermentation conditions, the highest yields obtained were 10.7 g of gamma-linolenic acid/kg of fermented product and 261.5 mg of beta-carotene/kg of fermented product. This yield of beta-carotene is the highest among the results published so far.

Production of Long Chain Fatty Alcohols Found in Bumblebee Pheromones by Yarrowia lipolytica.

Fatty alcohols (FA-OH) are aliphatic unbranched primary alcohols with a chain of four or more carbon atoms. Besides potential industrial applications, fatty alcohols have important biological functions as well. In nature, fatty alcohols are produced as a part of a mixture of pheromones in several insect species, such as moths, termites, bees, wasps, etc. In addition, FA-OHs have a potential for agricultural applications, for example, they may be used as a suitable substitute for commercial insecticides. The insecticides have several drawbacks associated with their preparation, and they exert a negative impact on the environment. Currently, pheromone components are prepared mainly through the catalytic hydrogenation of plant oils and petrochemicals, which is an unsustainable, ecologically unfriendly, and highly expensive process. The biotechnological production of the pheromone components using engineered microbial strains and through the expression of the enzymes participating in the biosynthesis of these components is a promising approach that ensures ecological sustenance as well. The present study was aimed at evaluating the production of FA-OHs in the oleaginous yeast, Yarrowia lipolytica, with different lengths of fatty-acyl chains by expressing the fatty acyl-CoA reductase (FAR) BlapFAR4 from B. lapidarius, producing C16:0-OH, C16:1Δ9-OH, and lower quantities of both C14:0-OH and C18:1Δ9-OH, and BlucFAR1 from B. lucorum, producing FA-OHs with a chain length of 18-26 carbon atoms, in this yeast. Among the different novel Y. lipolytica strains used in the present study, the best results were obtained with JMY7086, which carried several lipid metabolism modifications and expressed the BlucFAR1 gene under the control of a strong constitutive promoter 8UAS-pTEF. JMY7086 produced only saturated fatty alcohols with chain lengths from 18 to 24 carbon atoms. The highest titer and accumulation achieved were 166.6 mg/L and 15.6 mg/g DCW of fatty alcohols, respectively. Unlike JMY7086, the BlapFAR4-expressing strain JMY7090 produced only 16 carbon atom-long FA-OHs with a titer of 14.6 mg/L.

Overexpression of diacylglycerol acetyltransferase from Euonymus europaeus in Yarrowia lipolytica leads to the production of single-cell oil enriched with 3-acetyl-1,2-diacylglycerols.

The 3-acetyl-1,2-diacylglycerols (acTAGs) are the molecules that are structurally similar to triacylglycerols (TAGs). They are naturally produced by plants of the family Celastraceae and animals such as Cervus nippon and Eurosta solidaginis. The presence of acetate in the sn-3 position of the glycerol backbone confers advantages to these compounds, for example, lower viscosity and calorific value compared to classical TAGs. In this work, the gene EeDAcT, which encodes diacylglycerol acetyltransferase in a species of bush (Euonymus europaeus), was overexpressed in strains Po1d (capable of accumulating storage lipids) and JMY1877 (incapable of accumulating storage lipids) of Yarrowia lipolytica, to test the activity of the gene EeDAcT and the production of acTAGs in oleaginous and nonoleaginous genetic backgrounds. It was observed that both the strains containing the gene EeDAcT (YL33 and YL35 for Po1d and JMY1877 strains, respectively) produced acTAGs. The strain YL33 accumulated up to 20% intracellular lipids, 20% of which was acTAGs, and 40% was TAGs. On the other hand, the strain YL35, which showed interrupted TAGs accumulation, produced up to 10% acTAGs as the only storage lipid. Unfortunately, the quantity of acTAGs produced in YL35 was insignificant, as the overall lipid accumulated in the strain was not more than 4% of the biomass. The fatty acid profile of acTAGs produced by the YL33 strain was remarkably similar to TAGs, and both of these structures were rich in oleic (45%) and palmitic (25%) acids.

Mitochondrial Citrate Transport System in the Fungus Mucor circinelloides: Identification, Phylogenetic Analysis, and Expression Profiling During Growth and Lipid Accumulation.

The mitochondrial citrate transport system, composed of citrate and malate transporters (MTs), can regulate the citrate efflux from mitochondria to cytosol, and then citrate is cleaved into OAA and acetyl-CoA which can be used for fatty acid (FA) biosynthesis. However, in the fungus Mucor circinelloides the molecular mechanism of citrate efflux from the mitochondria by this system and its role in FA synthesis is unclear. In the present study, we have analyzed the genome of high lipid-producing strain WJ11 and the low lipid-producing strain CBS 277.49 to find the potential genes involving in this system. Five potential genes are present in the genome of WJ11. These genes encode one citrate transport protein (CT), one tricarboxylate carrier (TCT), one MT, and two 2-oxoglutarate:malate antiporters (SoDIT-a and SoDIT-b). However, the genome of CBS 277.49 contains the same set of genes, except for the presence of just one SoDIT. The proteins from WJ11 had similar properties as their counterparts in CBS 277.49. Moreover, phylogenetic analyses revealed the evolutionary relationship of these proteins and illuminated their typical motifs related to potential functions. Additionally, the expression of these genes was analyzed to predict the possible functions in lipid metabolism in M. circinelloides. This is the first study to report the in silico analysis of structures and functions of the mitochondrial citrate transport system in M. circinelloides. This work showed a new strategy for research for the selection of candidate genes for further detailed functional investigation of the mitochondrial citrate transport system in lipid accumulation.

Carotenoid-producing yeasts: Identification and Characteristics of Environmental Isolates with a Valuable Extracellular Enzymatic Activity.

Sixteen cold-adapted reddish-pigmented yeast strains were obtained from environmental samples. According to the PCR-based detection of classical yeast markers combined with phylogenetic studies, the yeasts belong mainly to the genera Rhodotorula, Sporobolomyces and Cystobasidium, all within the subphylum Pucciniomycotina. All strains produced carotenoids within a 0.25-10.33 mg/L range under non-optimized conditions. Noteworthily, among them, representatives of the Cystobasidium genus were found; of particular value are the strains C. laryngis and C. psychroaquaticum, poorly described in the literature to date. Interestingly, carotenoid production with representatives of Cystobasidium was improved 1.8- to 10-fold at reduced temperature. As expected, most of the isolated yeasts biosynthesized extracellular lipases, but within them also one proteolytic and four cellulolytic strains were revealed. We succeeded in isolating strain Cystofilobasidium macerans WUT145 with extraordinarily high cellulolytic activity at 22°C (66.23 ± 0.15 µmol/mg protein·min) that is described here for the first time. Consequently, a set of yeasts capable of producing both carotenoids and extracellular enzymes was identified. Taking into account those abilities, the strains might be applicable for a development of carotenoids production on an agro-industrial waste, e.g., lignocellulose.

Effect of Fungal Solid-State Fermented Product in Broiler Chicken Nutrition on Quality and Safety of Produced Breast Meat.

The aim of this work was to analyse the effect of addition of 10% (w/w) fermented bioproduct into commercial broiler feed on fatty acid profile, lipid oxidative stability, and sensory properties of chicken breast meat. The fermented bioproduct was prepared by fermentation of cornmeal by filamentous fungi Umbelopsis isabellina CCF 2412 in solid-state fermentation (SSF) process and the final bioproduct was enriched with gamma-linolenic acid and beta-carotene. In the experiment, 80 pieces of 1-day-old chickens COBB 500 were used. Half of them (control group) were fed only with commercial feed. Chickens of the experimental group were fed with commercial feed, and, from the 11th day of age until the time of slaughter (39th day), 10% of commercial feed was replaced with fermented bioproduct. Application of fermented bioproduct into commercial feed mixture positively influenced profile of fatty acids in breast meat. The amount of gamma-linolenic, alpha-linolenic, and oleic acids in fat of breast muscles was increased and n-6/n-3 ratio was significantly decreased. Profile and content of PUFAs did not change after thermal treatment of meat. Oxidative stability of fat and sensory properties of the meat during the storage (4°C, 7 days) of meat were not affected by fermented bioproduct.

Progress in the genetic engineering of cereals to produce essential polyunsaturated fatty acids.

Essential polyunsaturated fatty acids with more than two double bonds and length of carbon chain 18-22 must be taken in the diet to prevent diseases and imbalances caused by their deficiency. Terrestrial sources of polyunsaturated fatty acids are limited to only a few plant species whose large-scale cultivation is not possible and the production of their seeds and oil is ineffective. The complete biosynthetic pathway of fatty acids is known in organisms, including plants. After the first gene encoding the enzyme catalysing the initial steps of PUFA biosynthesis (ω-3 desaturase, Δ6-desaturase) were isolated, isolation of other genes encoding relevant enzymes of the PUFA pathway from different donor organisms followed. Genetic transformations of model plants by the desaturase- and elongase-encoding genes opened the way for the genetic engineering of oilseed crop species. Some of the developed transgenic plants produced PUFAs, including eicosapentaenoic and docosahexaenoic acids. Seed oils extracted from them were similar to fish oil. Tools of the synthetic biology can be applied in modifications of the PUFA pathway and also in overcoming of limitations when the gene and its expression product are absent in the pathway. Such progress in cereals (barley, wheat, maize) has been made only recently, when the first successful modifications of the ω-3 and ω-6 PUFA pathways succeeded. This review focuses on genetic modifications of the PUFA biosynthetic pathway in cereals in relation to the status reached in model plants and oilseed crops.

The Effect of Zn(II) Ions and Reactive Oxygen on the Uptake of Zinc and Production of Carotenoids by Selected Red Yeasts.

Three strains of red yeast Rhodosporidium kratochvilovae, Rhodotorula glutinis and Sporidiobolus salmonicolor were studied for their responses to the presence metal stress, oxidative stress and a combination of these stress factors. For all yeast strains, the production of ß-carotene increased in stress conditions. The combination of H2 O2 and Zn2+ significantly activated the pathways for the production of torularhodin in the strain R. glutinis (from 250 to 470 µg g-1 DCW) as well as ß-carotene (from 360 to 1100 µg g-1 DCW) and torulene (from 100 to 360 µg g-1 DCW) in Sp. salmonicolor. Strains of R. glutinis and Rh. kratochvilovae bound the majority of Zn(II) ions to the fibrillar part of the cell walls, whereas the strain Sp. salmonicolor bound them to both extracellular polymers and the fibrillar part of the cell walls. A decrease in the ability of yeasts to tolerate higher concentrations of Zn(II) in the presence of free radicals (hydrogen peroxide) was also found.

Lipid Peroxidation Process in Meat and Meat Products: A Comparison Study of Malondialdehyde Determination between Modified 2-Thiobarbituric Acid Spectrophotometric Method and Reverse-Phase High-Performance Liquid Chromatography.

The aim of this work was to compare the methods of malondialdehyde detection, as the main secondary product of the lipid peroxidation process, in meat and meat products. Malondialdehyde measurements were performed by two modified methods, the 2-thiobarbituric acid spectrophotometric method and the reverse-phase high-performance liquid chromatography in raw, mechanically-deboned chicken meat and in manufactured frankfurters. The malondialdehyde concentrations measured by the 2-thiobarbituric acid spectrophotometric method were found to be overestimated by more than 25% in raw meat and more than 27% in frankfurters in comparison to the results of reverse-phase high-performance liquid chromatography (p < 0.05). The achieved results showed that the presented modified reverse-phase high-performance liquid chromatography method was more applicable and more accurate for the quantification of malondialdehyde in samples of meat and meat products.