Applications of synthetic yeast consortia for the production of native and non-native chemicals.

The application of microbial consortia is a new approach in synthetic biology. Synthetic yeast consortia, simple or complex synthetic mixed cultures, have been used for the production of various metabolites. Cooperation between the members of a consortium and cross-feeding can be applied to create stable microbial communication. These consortia can: consume a variety of substrates, perform more complex functions, produce metabolites in high titer, rate, and yield (TRY), and show higher stability during industrial fermentations. Due to the new research context of synthetic consortia, few yeasts were used to build these consortia, including Saccharomyces cerevisiae, Pichia pastoris, and Yarrowia lipolytica. Here, application of the yeasts for design of synthetic microbial consortia and their advantages and bottlenecks for effective and robust production of valuable metabolites from bioresource, including: cellulose, xylose, glycerol and so on, have been reviewed. Key trends and challenges are also discussed for the future development of synthetic yeast consortia.

Microbial L-asparaginase as a promising enzyme for treatment of various cancers.

Metabolic differences between normal and cancerous cells have been used as a point of view for developing anticancer drugs. Some degrading enzymes of certain amino acids have been regarded to kill cancerous cells. L-Asparaginase (ASNase) has shown an excellent therapeutic response to asparagine-auxotrophic cancers such as acute lymphoblastic leukemia (ALL). Some bacteria, yeasts, molds, plants, and animals produce ASNase. Bacterial ASNases from Escherichia coli and Erwinia chrysanthemi are the FDA-approved drugs for ALL treatment. Here, we review new natural prokaryotic and eukaryotic sources of ASNases, recent advances to introduce improvement strategies for the production of recombinant ASNases as well as their chemical modifications, immobilization, nanoencapsulation, and in silico studies to increase efficiency and decrease side effects. Recent studies for application of ASNases to treatment of asparagine-auxotrophic cancers, especially solid cancers, have been reviewed. Furthermore, challenges and future perspectives are discussed for this promising therapeutic enzyme. KEY POINTS: • Review recent advances to introduce new sources of microbial L-asparaginases. • Review improvement strategies for the development of stable and non-toxic L-asparaginases. • Review microbial L-asparaginase application in various cancers' treatment.

Phenol biodegradation by immobilized Rhodococcus qingshengii isolated from coking effluent on Na-alginate and magnetic chitosan-alginate nanocomposite.

Phenol is a hazardous organic solvent to living organisms, even in its small amounts. In order to bioremediation of phenol from aqueous solution, a novel bacterial strain was isolated from coking wastewater, identified as Rhodococcus qingshengii based on 16S rRNA sequence analysis and named as strain Sahand110. The phenol-biodegrading capabilities of the free and immobilized cells of Sahand110 on the beads of Na-alginate (NA) and magnetic chitosan-alginate (MCA) nanocomposite were evaluated under different initial phenol concentrations (200, 400, 600, 800 and 1000 mg/L). Results illustrated that Sahand110 was able to grow and complete degrade phenol up to 600 mg/L, as the sole carbon and energy source. Immobilized cells of Sahand110 on NA and MCA were more competent than its free cells in degradation of high phenol concentrations, 100% of 1000 mg/L phenol within 96 h, indicating the improved tolerance and performance of the immobilized cells against phenol toxicity. Therefore, the immobilized Sahand110 on the studied beads, especially MCA bead regarding its suitable properties, has significant potential to enhanced bioremediation of phenol-rich wastewaters.

Metabolic engineering of Saccharomyces cerevisiae for production of ß-carotene from hydrophobic substrates.

ß-Carotene is a yellow-orange-red pigment used in food, cosmetics and pharmacy. There is no commercial yeast-based process for ß-carotene manufacturing. In this work, we engineered the baker's yeast Saccharomyces cerevisiae by expression of lipases and carotenogenic genes to enable the production of ß-carotene on hydrophobic substrates. First, the extracellular lipase (LIP2) and two cell-bound lipases (LIP7 and LIP8) from oleaginous yeast Yarrowia lipolytica were expressed either individually or in combination in S. cerevisiae. The engineered strains could grow on olive oil and triolein as the sole carbon source. The strain expressing all three lipases had ∼40% lipid content per dry weight. Next, we integrated the genes encoding ß-carotene biosynthetic pathway, crtI, crtYB and crtE from Xanthophyllomyces dendrorhous. The resulting engineered strain bearing the lipases and carotenogenic genes reached a titer of 477.9 mg/L ß-carotene in yeast peptone dextrose (YPD) medium supplemented with 1% (v/v) olive oil, which was 12-fold higher than an analogous strain without lipases. The highest ß-carotene content of 46.5 mg/g DCW was obtained in yeast nitrogen base (YNB) medium supplemented with 1% (v/v) olive oil. The study demonstrates the potential of applying lipases and hydrophobic substrate supplementation for the production of carotenoids in S. cerevisiae.

Advances in synthetic biology of oleaginous yeast Yarrowia lipolytica for producing non-native chemicals.

Oleaginous yeast Yarrowia lipolytica is an important industrial host for the production of enzymes, oils, fragrances, surfactants, cosmetics, and pharmaceuticals. More recently, improved synthetic biology tools have allowed more extensive engineering of this yeast species, which lead to the production of non-native metabolites. In this review, we summarize the recent advances of genome editing tools for Y. lipolytica, including the application of CRISPR/Cas9 system and discuss case studies, where Y. lipolytica was engineered to produce various non-native chemicals: short-chain fatty alcohols and alkanes as biofuels, polyunsaturated fatty acids for nutritional and pharmaceutical applications, polyhydroxyalkanoates and dicarboxylic acids as precursors for biodegradable plastics, carotenoid-type pigments for food and feed, and campesterol as a precursor for steroid drugs.

Modification of zeolite 4A for use as an adsorbent for glyphosate and as an antibacterial agent for water.

The aim of this work was to design a low cost adsorbent for efficient and selective removal of glyphosate from water at neutral pH conditions. For this purpose, zeolite 4A, a locally abundant and cheap mineral material, was ion-exchanged with Cu2+ to produce Cu-zeolite 4A. The FTIR results revealed that the modification has no important effect on chemical structure of zeolite 4A. After modification, highly crystalline zeolite 4A was converted to amorphous Cu-zeolite 4A according to XRD studies. The SEM images showed spherical-like particles with porous surfaces for Cu-zeolite 4A compared to cubic particles with smooth surfaces for zeolite 4A. Adsorption equilibrium data were well fitted with non-linear forms of Langmuir, Freundlich and Temkin isotherms. The maximum adsorption capacity for Cu-zeolite 4A was calculated to be 112.7 mg g-1 based on the Langmuir model. The adsorption of glyphosate by the modified adsorbent had fast kinetics fitted both pseudo-first-order and pseudo-second-order models. A mechanism based on chemical adsorption was proposed for the removal process. The modified adsorbent had a good selectivity to glyphosate over natural waters common cations and anions. It also showed desired regeneration ability as an important feature for practical uses. The potential use of the developed material as antibacterial agent for water disinfection filters was also investigated by MIC method. Relatively strong antibacterial activity was observed for Cu-zeolite 4A against Gram-positive and Gram-negative model bacteria while zeolite 4A had no antibacterial properties. No release of Cu2+ to aqueous solutions was detected as unique feature of the developed material.

Laccase production from sucrose by recombinant Yarrowia lipolytica and its application to decolorization of environmental pollutant dyes.

Laccases are used in decolorization and biodegradation of synthetic dyes, bioremediation of industrial wastewaters and delignification of lignocellulosic compounds. The aims of the present study were the optimization of a recombinant laccase production in Yarrowia lipolytica yeast using sucrose as a main carbon source, and the application of the resulting enzyme to decolorization of synthetic dyes, which are problematic environmental pollutants. Taguchi's experimental design method was employed to optimize medium compounds. Recombinant laccase production by Y. lipolytica YL4 strain increased to 900 U L-1 after optimization of sucrose, ammonium chloride, yeast extract and thiamine levels in the modified PPB medium. Furthermore, the production rate reached 6760 U L-1 in a 5 L bioreactor which represents 4.5- and 33.5-fold increases compared to cultures that were in shake-flask with optimized and primary media, respectively. The supernatant containing secreted recombinant laccase was applied for decolorization of seven dyes. The effects of pH, the amount of enzyme and incubation period were verified. The effect of incubation time on dye decolorization by recombinant laccase was important, which has an influence of greater extent than 90% after 48 h for all dyes. The Trametes versicolor laccase can be efficiently produced in Y. lipolytica and the recombinant enzyme has a considerable potential in the decolorization of pollutant synthetic dyes.

In silico and in vivo analysis of signal peptides effect on recombinant glucose oxidase production in nonconventional yeast Yarrowia lipolytica.

Signal peptide (SP) is an important factor and biobrick in the production and secretion of recombinant proteins. The aim of this study was in silico and in vivo analysis of SPs effect on the production of recombinant glucose oxidase (GOX) in Yarrowia lipolytica. Several in silico softwares, namely SignalP4, Signal-CF, Phobius, WolfPsort 0.2, SOLpro and ProtParam, were used to analyse the potential of 15 endogenous and exogenous SPs for the secretion of recombinant GOX in Y. lipolytica. According to in silico results, the SP of GOX was predicted as suitable in terms of high secretory potential and of protein solubility and stability which is chosen for in vivo analysis. The recombinant Y. lipolytica strain produced 280 U/L of extracellular GOX after 7 days in YPD medium. The results show that the SP of GOX can be applied to efficient production of extracellular heterologous proteins and metabolic engineering in Y. lipolytica.

Homology modeling and molecular docking studies to decrease glutamine affinity of Yarrowia lipolytica L-asparaginase.

L-Asparaginase is a key component in the treatment of leukemias and lymphomas. However, the glutamine affinity of this therapeutic enzyme is an off-target activity that causes several side effects. The modeling and molecular docking study of Yarrowia lipolytica L-asparaginase (YL-ASNase) to reduce its l-glutamine affinity and increase its stability was the aim of this study. Protein-ligand interactions of wild-type and different mutants of YL-ASNase against L-asparagine compared to l-glutamine were assessed using AutoDock Vina tools because the crystal structure of YL-ASNase does not exist in the protein data banks. The results showed that three mutants, T171S, T171S-N60A, and T171A-T223A, caused a considerable increase in L-asparagine affinity and a decrease in l-glutamine affinity as compared to the wild-type and other mutants. Then, molecular dynamics simulation and MM/GBSA free energy were applied to assess the stability of protein structure and its interaction with ligands. The three mutated proteins, especially T171S-N60A, had higher stability and interactions with L-asparagine than l-glutamine in comparison with the wild-type. The YL-ASNase mutants could be introduced as appropriate therapeutic candidates that might cause lower side effects. However, the functional properties of these mutated enzymes need to be confirmed by genetic manipulation and in vitro and in vivo studies.

Novel probe based on rhodamine B and quinoline as a naked-eye colorimetric probe for dual detection of nickel and hypochlorite ions.

This work demonstrates the design and straightforward syntheses of several novel probe-based on rhodamine B and 2-mercaptoquinoline-3-carbaldehydes as a naked-eye colorimetric probe, indicating a sensitive and selective recognition towards nickel (II) with a limit of detection 0.30 µmol L-1 (0.02 mg L-1). Further, by employing the oxidation property of hypochlorite (OCl-), this novel probe parallelly has been deployed to detect hypochlorite in laboratory conditions with a limit of detection of 0.19 µmol mL-1 and in living cells. Regarded to negligible cell toxicity toward mammalian cells, this probe has the potential to determine these analytes in in-vivo investigation and foodstuff samples.

Yarrowia lipolytica Bioprocess Development: From Flask to Bioreactor.

Yarrowia lipolytica produces a range of valuable biotechnological products from natural metabolites and enzymes to heterologous proteins. The production of these products is affected by medium composition and various environmental factors. Here we describe bioprocess development for a recombinant laccase production by Y. lipolytica. At first, response surface methodology (RSM), as a statistical technique for design of experiment (DOE), is used for the optimization of medium composition in flask level. Then, results of RSM are applied to increase laccase production in controlled conditions of the bioreactor.

Yarrowia lipolytica L-asparaginase inhibits the growth and migration of lung (A549) and breast (MCF7) cancer cells.

L-asparaginase is an enzyme capable of hydrolyzing the asparagine to aspartic acid and ammonia. L-asparaginase is widely used in the treatment of acute lymphoblastic leukemia (ALL) and other cancers. Here, for the first time, the effects of a novel yeast L-asparaginase from Yarrowia lipolytica were studied on human lung (A549) and breast cancer (MCF7) cell lines as the solid cancer cell lines in terms of cell growth and metastasis inhibition. Functional analysis showed the L-asparagine deprivation mediated anti-proliferation effects by apoptosis induction and changes in the expression of target genes involved in apoptosis and migration pathways. The qRT-PCR analysis showed the higher expression levels of pro-apoptosis genes, including Bax, P53, caspase 3, caspase 8, and down-regulation of Bcl-2 anti-apoptotic gene in treated cells. On the other hand, there was no increase in ROS production in the treated cells. However, L-asparaginase treatment was able to significantly induce autophagy activation in A549 cells. Besides, wound healing assay showed that L-asparaginase could considerably inhibit the migration of A549 and MCF7 cells. Taken together, our results suggested that Yarrowia lipolytica L-asparaginase might be considered for enzyme therapy against breast and lung cancers.

The effect of Yarrowia lipolytical-asparaginase on apoptosis induction and inhibition of growth in Burkitt's lymphoma Raji and acute lymphoblastic leukemia MOLT-4 cells.

l-Asparaginase (l-asparagine amidohydrolase; E.C.3.5.1.1) as an anticancer agent is used to treat acute lymphocytic leukemia (ALL), Human Burkitt's lymphoma and non-Hodgkin's lymphoma. The commercial asparaginases are obtained from bacteria Erwinia chrysanthemi and Escherichia coli now which had many side effects. In this study, the effects of a novel l-asparaginase from yeast Yarrowia lipolytica was investigated on human ALL and Burkitt's lymphoma cell lines. The l-asparaginase causes metabolic stress, cytotoxicity, and apoptosis due to the arrest of the G0 cell cycle, the activation of caspase-3 and the modulation of mitochondrial membrane integrity. The RT-PCR analysis showed a significant increase in the pro-apoptosis genes such as Bax, Caspase-3, Caspase-8, Caspase-9 and p53 (P < 0.05) while the anti-apoptotic marker Bcl-2 was significantly decreased (P < 0.05). Furthermore, Y. lipolytical-asparaginase causes autophagy and increased ROS. The l-asparaginase has cytotoxic and anticancer effects higher than commercial asparaginase. In conclusion, Y. lipolytical-asparaginase shows interesting anticancer activity and it can be introduced as a new eukaryotic and therapeutic agent and strategy for ALL and Burkitt's lymphoma treatment after the in vivo and clinical experiments.

Effect of plant oils upon lipase and citric acid production in Yarrowia lipolytica yeast.

The nonconventional yeast Yarrowia lipolytica degrades very efficiently hydrophobic substrates to produce organic acids, single-cell oil, lipases, and so forth. The aim of this study was to investigate the biochemical behavior and simultaneous production of valuable metabolites such as lipase, citric acid (CA), and single-cell protein (SCP) by Yarrowia lipolytica DSM 3286 grown on various plant oils as sole carbon source. Among tested plant oils, olive oil proved to be the best medium for lipase and CA production. The Y. lipolytica DSM 3286 produced 34.6 +/- 0.1 U/mL of lipase and also CA and SCP as by-product on olive oil medium supplemented with yeast extract. Urea, as organic nitrogen, was the best nitrogen source for CA production. The results of this study suggest that the two biotechnologically valuable products, lipase and CA, could be produced simultaneously by this strain using renewable low-cost substrates such as plant oils in one procedure.

Production and structural modeling of a novel asparaginase in Yarrowia lipolytica.

Asparaginase catalyzes the conversion of asparagine into aspartic acid and ammonia. The enzyme has various industrial applications and it is considered as an anticancer drug for treatment of certain leukemias. In the current study, production of asparaginase was investigated by Yarrowia lipolytica as well as optimized its production and determined its molecular characteristics by in silico analysis. Y. lipolytica DSM3286 produced 17.14 U/ml of asparaginase in flask culture. Optimization of asparaginase production was done by response surface methodology and the enzyme production increases up to 102.85 U/ml. The enzyme production reached 210 U/ml in a bioreactor which is 12-fold more than flask culture containing non-optimized medium. Asparaginase gene of Y. lipolytica was identified and isolated on the basis of comparison with asparaginase gene sequences of other microorganisms. The gene has 981 nucleotides and its protein has 326 amino acids. According to in silico analysis, the secondary structure of the enzyme is composed of 9 α-helixes and 11 ß-sheets. Y. lipolytica produces type II asparaginase with high affinity for asparagine which is a suitable eukaryotic asparaginase for treatment of hematopoietic cancers. Hence, Y. lipolytica could be recommended as a new eukaryotic microbial source for the production of this important therapeutic enzyme.

Noncovalent Immobilization of Yarrowia lipolytica Lipase on Dendritic-Like Amino Acid-Functionalized Silica Nanoparticles.

Immobilization of enzymes is a promising approach for the cost-effective application of enzymes. Among others, noncovalent but unleachable approaches for immobilization are one of the most favorable and crucial approaches. Herein, silica nanoparticles are modified by (3-aminopropyl)triethoxysilane (APTES) to generate amino-functionalized silica nanoparticles. Then, the amine functionalities are converted to bifunctional amino acid via post-modification that has zwitterionic properties. This nanostructure with the new functional theme is employed to immobilize Yarrowia lipolytica lipase at room temperature with no further post-modification or cross-linking. This immobilization method is further compared with the metal chelate-based immobilization approach on the same support. The biocatalytic activity of the immobilized lipase is examined under various conditions. The encapsulation of lipase through amino acid-functionalized silica nanoparticles exhibited enhanced stability for the immobilized lipase at higher temperatures and unneutral pHs.

Interaction of Yarrowia lipolytica lipase with dithiocarbamate modified magnetic carbon Fe3O4@C-NHCS2H core-shell nanoparticles.

Fe3O4@C core-shell nanoparticles were modified by (3-aminopropyl)triethoxysilane (APTES) to generate amine functionality in the surface. Then, the amine functional groups were converted to dithiocarbamate via post-modification with carbon disulfide. This nanostructure with new functional property was used to immobilize lipase (obtained from Yarrowia lipolytica U6). Biocatalytic activity of the Fe3O4@C-NHCS-LIP was studied in this project. The interaction of lipase and support though dithiocarbamate binder was examined in the hydrolysis of p-nitrophenyl laurate. In this paper, support showed a unique feature in the immobilization of lipase by maintaining the lipase activity, raising the stability of lipase, and reusability.

Production of Laccase by Recombinant Yarrowia lipolytica from Molasses: Bioprocess Development Using Statistical Modeling and Increase Productivity in Shake-Flask and Bioreactor Cultures.

Laccases are used in numerous applications, from green degradation of various xenobiotic compounds, waste detoxification, textile dye bleaching, and delignification of lignocellulose materials to biofuel production. In this study, the recombinant Yarrowia lipolytica YL4 strain carrying the white-rot fungus Trametes versicolor laccase IIIb gene was used for laccase production from beet molasses as an agro-industrial residue. Response surface methodology was used to statistical optimization of the production of laccase by Y. lipolytica using an industrial medium containing molasses which allows a six times increase in laccase activity compared to primary medium contains glucose after 144 h. In bioreactor cultivation after 48 h, laccase production reached to 3.7- and 22.5-fold more than optimized and primary media in shake-flask cultures, respectively. Laccase productivity in bioreactor (0.0937 U/h) was higher than shake-flask culture (0.0084 U/h). The present study provides valuable information about statistical optimization of bioprocess development for cost-effective production of laccase and other heterologous proteins in Y. lipolytica from beet molasses as sole carbon source, thus allowing the valorization and decreasing environmental pollution of this agro-industrial waste.

Expression and Characterization of Glucose Oxidase from Aspergillus niger in Yarrowia lipolytica.

Glucose oxidase (GOX) is currently used in clinical, pharmaceutical, food and chemical industries. The aim of this study was expression and characterization of Aspergillus niger glucose oxidase gene in the yeast Yarrowia lipolytica. For the first time, the GOX gene of A. niger was successfully expressed in Y. lipolytica using a mono-integrative vector containing strong hybrid promoter and secretion signal. The highest total glucose oxidase activity was 370 U/L after 7 days of cultivation. An innovative method was used to cell wall disruption in current study, and it could be recommended to use for efficiently cell wall disruption of Y. lipolytica. Optimum pH and temperature for recombinant GOX activity were 5.5 and 37 °C, respectively. A single band with a molecular weight of 80 kDa similar to the native and pure form of A. niger GOX was observed for the recombinant GOX in SDS-PAGE analysis. Y. lipolytica is a suitable and efficient eukaryotic expression system to production of recombinant GOX in compered with other yeast expression systems and could be used to production of pure form of GOX for industrial applications.