Enhanced Production of Erythritol from Glucose by the Newly Obtained UV Mutant Yarrowia lipolytica K1UV15.

Erythritol is a polyol with a sweet taste but low energy value. Thanks to its valuable properties, as well as growing social awareness and nutritional trends, its popularity is growing rapidly. The aim of this study was to increase the effectiveness of erythritol production from glucose using new UV mutants of the yeast Yarrowia lipolytica obtained in the Wratislavia K1 strain. The ability of the new strains to biosynthesize erythritol and utilize this polyol was examined in shake-flask cultures and fed-batch processes conducted in a stirred tank reactor with a total glucose concentration of 300 and 400 g/L. The Wratislavia K1 strain produced erythritol most efficiently (97.5 g/L; 192 h) at an initial glucose concentration of 250 g/L (total: 300 g/L). New strains were assessed under such conditions, and it was noted that the highest erythritol concentration (145 g/L; 183 h) was produced by the K1UV15 strain. A significant improvement in the erythritol biosynthesis efficiency (148 g/L; 150 h) was achieved upon the increase in (NH4)2SO4 to 3.6 g/L. Further, in the culture with such a concentration of the nitrogen source and increased total glucose level (400 g/L), the K1UV15 strain produced 226 g/L of erythritol within 281 h.

Determination of Bioactive Compound Kynurenic Acid in Linum usitatissimum L.

Kynurenic acid (KYNA) is a bioactive compound exhibiting multiple actions and positive effects on human health due to its antioxidant, anti-inflammatory and neuroprotective properties. KYNA has been found to have a beneficial effect on wound healing and the prevention of scarring. Despite notable progress in the research focused on KYNA observed during the last 10 years, KYNA's presence in flax (Linum usitatissimum L.) has not been proven to date. In the present study, parts of flax plants were analysed for KYNA synthesis. Moreover, eight different cultivars of flax seeds were tested for the presence of KYNA, resulting in a maximum of 0.432 µg/g FW in the seeds of the cultivar Jan. The level of KYNA was also tested in the stems and roots of two selected flax cultivars: an oily cultivar (Linola) and a fibrous cultivar (Nike). The exposure of plants to the KYNA precursors tryptophan and kynurenine resulted in higher levels of KYNA accumulation in flax shoots and roots. Thus, the obtained results indicate that KYNA might be synthesized in flax. The highest amount of KYNA (295.9 µg/g dry weight [DW]) was detected in flax roots derived from plants grown in tissue cultures supplemented with tryptophan. A spectroscopic analysis of KYNA was performed using the FTIR/ATR method. It was found that, in tested samples, the characteristic KYNA vibration bands overlap with the bands corresponding to the vibrations of biopolymers (especially pectin and cellulose) present in flax plants and fibres.

Application of New Yarrowia lipolytica Transformants in Production of Citrates and Erythritol from Glycerol.

Citric acid and erythritol are obtained on an industrial scale using biotechnological methods. Due to the growing market demand for these products, research is underway to improve the process economics by introducing new microorganisms, in particular of the species Yarrowia lipolytica. The aim of this study was to evaluate transformants of Y. lipolytica for growth and ability to overproduce citric acids and erythritol from glycerol. The transformants were constructed by overexpressing glycerol kinase, methylcitrate synthase and mitochondrial succinate-fumarate transporter in the mutant Wratislavia 1.31. Next, strains were assessed for biosynthesis of citrate (pH 5.5; nitrogen limitation) and erythritol (pH 3.0; high osmotic pressure) from glycerol. Regardless of culture conditions strains, 1.31.GUT1/6 and 1.31.GUT1/6.CIT1/3 exhibited high rates of substrate utilization. Under conditions favoring citrate biosynthesis, both strains produced several percent more citrates, accompanied by higher erythritol production compared to the parental strain. During erythritol biosynthesis, the strain 1.31.GUT1/6.CIT1/3.E34672g obtained as a result of co-expression of all three genes stood out, producing 84.0 g/L of erythritol with yield and productivity of 0.54 g/g and 0.72 g/Lh, respectively, which places it in the group of the highest-ranked producers of erythritol among Y. lipolytica species.

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.

Sugar Alcohol Sweetener Production by Yarrowia lipolytica Grown in Media Containing Glycerol.

Most of the world's annual production of mannitol is by chemical means, but, due to increasing demand for natural sweeteners, alternative production methods are being sought. The aim of the study was to screen Yarrowia lipolytica yeast strains and select culture conditions for the efficient and selective biosynthesis of mannitol from glycerol. From 21 strains examined in the shake-flask culture for mannitol biosynthesis from glycerol (100 g/L), three strains were selected-S2, S3, and S4-and further evaluated in batch bioreactor cultures with technical and raw glycerol (150 g/L). The best production parameters were observed for strain S3, which additionally was found to be the most resistant to NaCl concentration. Next, strain S3 was examined in batch culture with regard to the initial glycerol concentration (from 50 to 250 g/L). It was found that the substrate concentrations of 50 and 75 g/L resulted in the highest mannitol selectivity, about 70%. The fed-batch culture system proposed in this paper (performed in two variants in which glycerol was dosed in four portions of about 50 or 75 g/L) resulted in increased mannitol production, up to 78.5 g/L.

High value-added products derived from crude glycerol via microbial fermentation using Yarrowia clade yeast.

BACKGROUND: Contemporary biotechnology focuses on many problems related to the functioning of developed societies. Many of these problems are related to health, especially with the rapidly rising numbers of people suffering from civilization diseases, such as obesity or diabetes. One factor contributing to the development of these diseases is the high consumption of sucrose. A very promising substitute for this sugar has emerged: the polyhydroxy alcohols, characterized by low caloric value and sufficient sweetness to replace table sugar in food production. RESULTS: In the current study, yeast belonging to the Yarrowia clade were tested for erythritol, mannitol and arabitol production using crude glycerol from the biodiesel and soap industries as carbon sources. Out of the 13 tested species, Yarrowia divulgata and Candida oslonensis turned out to be particularly efficient polyol producers. Both species produced large amounts of these compounds from both soap-derived glycerol (59.8-62.7 g dm-3) and biodiesel-derived glycerol (76.8-79.5 g dm-3). However, it is equally important that the protein and lipid content of the biomass (around 30% protein and 12% lipid) obtained after the processes is high enough to use this yeast in the production of animal feed. CONCLUSIONS: The use of waste glycerol for the production of polyols as well as utilization of the biomass obtained after the process for the production of feed are part of the development of modern waste-free technologies.

Application of a New Engineered Strain of Yarrowia lipolytica for Effective Production of Calcium Ketoglutarate Dietary Supplements.

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 products with desired nutritional and health-beneficial characteristics was demonstrated: (1) calcium α-ketoglutarate (CaKGA) with purity of 89.9% obtained by precipitation of KGA with CaCO3, (2) yeast biomass with very good nutritional properties, (3) fixed biomass-CaKGA preparation containing 87.2 µg/g of kynurenic acid, which increases the health-promoting value of the product.

Sustainable Surfactin Production by Bacillus subtilis Using Crude Glycerol from Different Wastes.

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). Furthermore, the surfactin produced in the study exhibited good stability in a wide range of pH, salinity and temperatures, suggesting its potential for several applications in biotechnology.

Effect of the Esterification of Starch with a Mixture of Carboxylic Acids from Yarrowia lipolitica Fermentation Broth on Its Selected Properties.

Potato starch was esterified with carboxylic acids contained in the fermentation broth from Yarrowia lipolitica yeast production. Various acid concentrations and various roasting temperatures were used to determine effects of process conditions on ester properties, including the number of acid residues attached to starch chains, starch susceptibility to amylolysis, and thermal characteristics of starch phase transitions. Study results demonstrated the effect of both the composition and the dose of the fermentation broth and of roasting temperature of starch on the number of acid residues attached to starch chains. Citric acid was more susceptible to esterification with starch (DS = 5.65%) compared to the α-ketoglutaric acid (DS = 0.12%). In the case of the latter, a higher degree of substitution was determined in the esters produced at higher roasting temperatures. The lowest digestibility (RS = 20%) was demonstrated for the starch esters with the highest degree of substitution with citric acid, whereas all starch esters showed decreased values of the thermal characteristics of pasting.

High-yield expression of extracellular lipase from Yarrowia lipolytica and its interactions with lipopeptide biosurfactants: A biophysical approach.

The unconventional yeast Yarrowia lipolytica is known as a producer of extracellular lipases. Here we overexpressed extracellular lipase (YlLip2) in yeast strain Y. lipolytica AJD ΔXΔA-Lip2 harboring the overexpression cassette of the YALI0A20350 gene under the strong hybrid promoter UAS1B16-TEF. To maintain a high level of YlLip2 production, two extracellular proteases of Y. lipolytica, AEPp and AXPp, were deleted. The purified recombinant YlLip2 presented optimal catalytic activities at 37 °C and pH 8.0. The effect of two lipopeptide biosurfactants, i.e., amphisin produced by Pseudomonas fluorescens DSS73 and viscosinamide secreted by P. fluorescens DR54, on the conformation and activity of YlLip2 was evaluated using spectral methods, surface tension, and the enzyme activity assay. YlLip2 demonstrated high tolerance of the tested biosurfactants and had greater activity retention after incubation with both biosurfactants. Finally, we observed that intrinsic fluorescence intensity of YlLip2 decreased significantly with increasing lipopeptides concentration ranging from 2.5 to 60 µM. Our results showed that both biosurfactants improve enzymatic activity of YlLip2 and might suggest better interaction of the substrate with the active site. These favorable characteristics make YlLip2 a prospective additive in the pharmaceutical, food, cosmetic, and detergent industries.

An efficient method for production of kynurenic acid by Yarrowia lipolytica.

Kynurenic acid (KYNA) is a compound derived from the tryptophan catabolic pathway. Antioxidant and neuroprotective properties have been confirmed for KYNA, which makes it an interesting and important metabolite of biomedical significance. In the present study, the yeast Yarrowia lipolytica was tested for KYNA biosynthesis. The results showed that Y. lipolytica strain S12 is able to produce KYNA in high concentrations (up to 21.38 µg/ml in culture broth and 494.16 µg/g cell dry weight in biomass) in optimized conditions in a medium supplemented with tryptophan. Different conditions of culture growth, including the source of carbon, its concentration and pH value of the medium, as well as the influence of an inhibitor or precursor of KYNA synthesis, were analysed. The obtained data confirmed the presence of KYNA metabolic pathway in the investigated yeast. To our best knowledge, this is the first study that reports KYNA production in the yeast Y. lipolytica in submerged fermentation.

Organic Acids of the Microbiological Post-Culture Medium as Substrates to be Used for Starch Modification.

This study aimed to identify the feasibility of producing highly-substituted starch esters via thermal modification of starch using a post-culture medium of Yarrowia lipolitica yeast. This manuscript describes a successful attempt at potato starch modification with a post-culture medium of Yarrowia lipolitica yeast with different concentrations of organic acids. Starch preparations produced by roasting (130 °C) and these produced by starch reaction with a synthetic acid mixture were compared in terms of the types and number of acid residues esterified with starch. The effectiveness of starch esterification was found to depend on medium composition and to be higher upon the use of a synthetic acid mixture. In addition, a higher reactivity with starch was demonstrated for citric acid than for α-ketoglutaric acid. The highly-substituted starch esters formed as a result of potato starch modification with post-culture medium were characterized by decreased values of thermal parameters of pasting characteristics, determined with a differential scanning calorimeter (DSC), and by compromised resistance to amylolysis. The intensity of these changes increased along with an increasing total percentage of starch ester substitution.

Production of high titer of citric acid from inulin.

BACKGROUND: Citric acid is considered as the most economically feasible product of microbiological production, therefore studies on cheap and renewable raw materials for its production are highly desirable. In this study citric acid was synthesized by genetically engineered strains of Yarrowia lipolytica from widely available, renewable polysaccharide - inulin. Hydrolysis of inulin by the Y. lipolytica strains was established by expressing the inulinase gene (INU1 gene; GenBank: X57202.1) with its native secretion signal sequence was amplified from genomic DNA from Kluyveromyces marxianus CBS6432. To ensure the maximum citric acid titer, the optimal cultivation strategy-repeated-batch culture was applied. RESULTS: The strain Y. lipolytica AWG7 INU 8 secreted more than 200 g dm- 3 of citric acid during repeated-batch culture on inulin, with a productivity of 0.51 g dm- 3 h- 1 and a yield of 0.85 g g- 1. CONCLUSIONS: The citric acid titer obtained in the proposed process is the highest value reported in the literature for Yarrowia yeast. The obtained results suggest that citric acid production from inulin by engineered Y. lipolytica may be a very promising technology for industrial citric acid production.

Aseptic production of citric and isocitric acid from crude glycerol by genetically modified Yarrowia lipolytica.

The unconventional yeast Yarrowia lipolytica is known for its capacity to produce citric or isocitric acid from glycerol. In this study a reduction of production cost was achieved by using cheap crude glycerol and conducting the production at pH 3 to prevent bacterial contamination. In this study a Y. lipolytica strain overexpressing Gut1 and Gut2 was used. For the modified strain, crude glycerol proved to be an excellent substrate for production of citric/isocitric acids in aseptic conditions, as the final concentration of these compounds reached 75.9 ±â€¯1.8 g L-1 after 7 days of batch production. Interestingly, the concentration of isocitric acid was 42.5 ±â€¯2.4 g L-1, which is one of the highest concentrations of isocitric acid obtained from a waste substrate. In summary, these data show that organic acids can be efficiently produced by the yeast Y. lipolytica from crude glycerol without any prior purification in aseptic conditions.

Effect of mcl-PHA synthesis in flax on plant mechanical properties and cell wall composition.

The high demand for new biomaterials makes synthesis of polyhydroxyalkanoates (PHA) in plants an interesting and desirable achievement. Production of polymers in plants is an example of application of biotechnology for improving the properties of plants, e.g. industrial properties, but it can also provide knowledge about plant physiology and metabolism. The subject of the present study was an industrially important plant: flax, Linum usitatissimum L., of a fibre cultivar (cv Nike). In the study the gene encoding PHA synthase from Pseudomonas aeruginosa, fused to a peroxisomal targeting signal, was expressed in flax plants with the aim of modifying the mechanical properties of plants. Medium-chain-length (mcl) hydroxy acids in flax plants from tissue cultures were detected by GC-FID and FTIR method. The introduced changes did not affect fatty acid content and composition in generated flax plants. Since mcl-PHA are known as elastomers, the mechanical properties of created plants were examined. Modified plants showed increases in the values of all measured parameters (except strain at break evaluated for one modified line). The largest increase was noted for tensile stiffness, which was 2- to 3-fold higher than in wild-type plants. The values estimated for another parameter, Young's modulus, was almost at the same level in generated flax plants, and they were about 2.7-fold higher when compared to unmodified plants. The created plants also exhibited up to about 2.4-fold higher tensile strength. The observed changes were accompanied by alterations in the expression of selected genes, related to cell wall metabolism in line with the highest expression of phaC1 gene. Biochemical data were confirmed by spectroscopic methods, which also revealed that crystallinity index values of cellulose in modified flax plants were increased in comparison to wild-type flax plants and correlated with biomechanical properties of plants.

Degradation of plastics and plastic-degrading bacteria in cold marine habitats.

Synthetic plastics present in everyday materials constitute the main anthropogenic debris entering the Earth's oceans. The oceans provide important and valuable resources such as food, energy, and water. They are also the main way of international trade and the main stabilizer of the climate. Hence, changes in the marine ecosystem caused by anthropogenic influences such as plastic pollution can have a dramatic impact on a global scale. Although the problem of plastics still remains unsolved, different ways are being considered to reduce their impact on the environment. One of them is to use microorganisms capable of degradation of plastic. A particularly interesting area is the application of microorganisms isolated from cold regions in view of their unique characteristics. Nevertheless, the interactions between plastic and microorganisms are still poorly known. Here, we present a review of current knowledge on plastic degradation and plastic-microorganism interactions in cold marine habitats. Moreover, we highlight the advantages of microorganisms isolated from this environment for eliminating plastic waste from ecosystems.

Recent advances in biological production of erythritol.

Erythritol is a natural sweetener commonly used in the food and pharmaceutical industries. Produced by microorganisms as an osmoprotectant, it is an ideal sucrose substitute for diabetics or overweight persons due to its almost zero calorie content. Currently, erythritol is produced on an industrial scale through the fermentation of sugars by some yeasts, such as Moniliella sp. However, the popularity of erythritol as a sweetener is still small because of its high retail price. This creates an opportunity for further process improvement. Recent years have brought the rapid development of erythritol biosynthesis methods from the low-cost substrates, and a better understanding of the metabolic pathways leading to erythritol synthesis. The yeast Yarrowia lipolytica emerges as an organism effectively producing erythritol from pure or crude glycerol. Moreover, novel erythritol producing organisms and substrates may be taken into considerations due to metabolic engineering. This review focuses on the modification of erythritol production to use low-cost substrates and metabolic engineering of the microorganisms in order to improve yield and productivity.

EUF1 - a newly identified gene involved in erythritol utilization in Yarrowia lipolytica.

The gene YALI0F01562g was identified as an important factor involved in erythritol catabolism of the unconventional yeast Yarrowia lipolytica. Its putative role was identified for the first time by comparative analysis of four Y. lipolytica strains: A-101.1.31, Wratislavia K1, MK1 and AMM. The presence of a mutation that seriously damaged the gene corresponded to inability of the strain Wratislavia K1 to utilize erythritol. RT-PCR analysis of the strain MK1 demonstrated a significant increase in YALI0F01562g expression during growth on erythritol. Further studies involving deletion and overexpression of the selected gene showed that it is indeed essential for efficient erythritol assimilation. The deletion strain Y. lipolytica AMM∆euf1 was almost unable to grow on erythritol as the sole carbon source. When the strain was applied in the process of erythritol production from glycerol, the amount of erythritol remained constant after reaching the maximal concentration. Analysis of the YALI0F01562g gene sequence revealed the presence of domains characteristic for transcription factors. Therefore we suggest naming the studied gene Erythritol Utilization Factor - EUF1.

Isolation and characterization of Arctic microorganisms decomposing bioplastics.

The increasing amount of plastic waste causes significant environmental pollution. In this study, screening of Arctic microorganisms which are able to degrade bioplastics was performed. In total, 313 microorganisms were isolated from 52 soil samples from the Arctic region (Spitsbergen). Among the isolated microorganisms, 121 (38.66%) showed biodegradation activity. The ability of clear zone formation on emulsified poly(butylene succinate-co-adipate) (PBSA) was observed for 116 microorganisms (95.87%), on poly(butylene succinate) (PBS) for 73 microorganisms (60.33%), and on poly(ɛ-caprolactone) (PCL) for 102 microorganisms (84.3%). Moreover, the growth of microorganisms on poly(lactic acid) (PLA) agar plates was observed for 56 microorganisms (46.28%). Based on the 16S rRNA sequence, 10 bacterial strains which showed the highest ability for biodegradation were identified as species belonging to Pseudomonas sp. and Rhodococcus sp. The isolated fungal strains were tested for polycaprolactone films and commercial corn and potato starch bags degradation under laboratory conditions. Strains 16G (based on the analysis of a partial 18S rRNA sequence, identified as Clonostachys rosea) and 16H (identified as Trichoderma sp.) showed the highest capability for biodegradation. A particularly high capability for biodegradation was observed for the strain Clonostachys rosea, which showed 100% degradation of starch films and 52.91% degradation of PCL films in a 30-day shake flask experiment. The main advantage of the microorganisms isolated from Arctic environment is the ability to grow at low temperature and efficient biodegradation under this condition. The data suggest that C. rosea can be used in natural and laboratory conditions for degradations of bioplastics.

Polyol production from waste materials by genetically modified Yarrowia lipolytica.

Sugar alcohols (polyols) are sweeteners with many industrial applications. In this study, a fermentation process of polyol production based on waste substrates - raw industrial molasses and crude glycerol - was tested. The yeast strain Yarrowia lipolytica Wratislavia K1 was genetically modified by overexpression of the Saccharomyces cerevisiae SUC2 gene and overexpression of the native GUT1 gene. This process allowed for sucrose utilization and rapid glycerol assimilation by the engineered strain. In this study, the obtained strain AIB pAD-UTGut1 produced 100.65±3.75g/l of polyols, with productivity of 1.09±0.9g/lh and yield of 0.67±0.2g/g. This is the first study describing efficient polyol production by the modified Y. lipolytica strain from industrial raw molasses and crude glycerol. By process optimization, we established conditions for abundant polyol synthesis from low-value substrates.