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1.
Toxins (Basel) ; 13(11)2021 10 24.
Article in English | MEDLINE | ID: mdl-34822538

ABSTRACT

Multicopper oxidases (MCOs) are a diverse group of enzymes that could catalyze the oxidation of different xenobiotic compounds, with simultaneous reduction in oxygen to water. Aside from laccase, one member of the MCO superfamily has shown great potential in the biodegradation of mycotoxins; however, the mycotoxin degradation ability of other MCOs is uncertain. In this study, a novel MCO-encoding gene, StMCO, from Streptomyces thermocarboxydus, was identified, cloned, and heterologously expressed in Escherichia coli. The purified recombinant StMCO exhibited the characteristic blue color and bivalent copper ion-dependent enzyme activity. It was capable of oxidizing the model substrate ABTS, phenolic compound DMP, and azo dye RB5. Notably, StMCO could directly degrade aflatoxin B1 (AFB1) and zearalenone (ZEN) in the absence of mediators. Meanwhile, the presence of various lignin unit-derived natural mediators or ABTS could significantly accelerate the degradation of AFB1 and ZEN by StMCO. Furthermore, the biological toxicities of their corresponding degradation products, AFQ1 and 13-OH-ZEN-quinone, were remarkably decreased. Our findings suggested that efficient degradation of mycotoxins with mediators might be a common feature of the MCOs superfamily. In summary, the unique properties of MCOs make them good candidates for degrading multiple major mycotoxins in contaminated feed and food.


Subject(s)
Aflatoxin B1/metabolism , Bacterial Proteins/metabolism , Oxidoreductases/metabolism , Streptomyces/enzymology , Zearalenone/metabolism , Bacterial Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Laccase/metabolism , Organisms, Genetically Modified/genetics , Organisms, Genetically Modified/metabolism
2.
Front Immunol ; 12: 647987, 2021.
Article in English | MEDLINE | ID: mdl-34248935

ABSTRACT

Cutaneous leishmaniasis caused by L. braziliensis induces a pronounced Th1 inflammatory response characterized by IFN-γ production. Even in the absence of parasites, lesions result from a severe inflammatory response in which inflammatory cytokines play an important role. Different approaches have been used to evaluate the therapeutic potential of orally administrated heat shock proteins (Hsp). These proteins are evolutionarily preserved from bacteria to humans, highly expressed under inflammatory conditions and described as immunodominant antigens. Tolerance induced by the oral administration of Hsp65 is capable of suppressing inflammation and inducing differentiation in regulatory cells, and has been successfully demonstrated in several experimental models of autoimmune and inflammatory diseases. We initially administered recombinant Lactococcus lactis (L. lactis) prior to infection as a proof of concept, in order to verify its immunomodulatory potential in the inflammatory response arising from L. braziliensis. Using this experimental approach, we demonstrated that the oral administration of a recombinant L. lactis strain, which produces and secretes Hsp65 from Mycobacterium leprae directly into the gut, mitigated the effects of inflammation caused by L. braziliensis infection in association or not with PAM 3CSK4 (N-α-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine, a TLR2 agonist). This was evidenced by the production of anti-inflammatory cytokines and the expansion of regulatory T cells in the draining lymph nodes of BALB/c mice. Our in vitro experimental results suggest that IL-10, TLR-2 and LAP are important immunomodulators in L. braziliensis infection. In addition, recombinant L. lactis administered 4 weeks after infection was observed to decrease lesion size, as well as the number of parasites, and produced a higher IL-10 production and decrease IFN-γ secretion. Together, these results indicate that Hsp65-producing L. lactis can be considered as an alternative candidate for treatment in both autoimmune diseases, as well as in chronic infections that cause inflammatory disease.


Subject(s)
Bacterial Proteins/administration & dosage , Bacterial Proteins/metabolism , Chaperonin 60/administration & dosage , Chaperonin 60/metabolism , Immune Tolerance/drug effects , Lactococcus lactis/metabolism , Leishmania braziliensis/drug effects , Leishmaniasis, Cutaneous/drug therapy , Mycobacterium leprae/enzymology , Administration, Oral , Animals , Bacterial Proteins/genetics , Chaperonin 60/genetics , Cytokines/metabolism , Female , Inflammation/drug therapy , Inflammation/immunology , Lactococcus lactis/genetics , Leishmaniasis, Cutaneous/immunology , Leishmaniasis, Cutaneous/parasitology , Mice , Mice, Inbred BALB C , Organisms, Genetically Modified/metabolism , Signal Transduction/drug effects , Signal Transduction/immunology , T-Lymphocytes, Regulatory/immunology
3.
World J Microbiol Biotechnol ; 37(5): 81, 2021 Apr 12.
Article in English | MEDLINE | ID: mdl-33843020

ABSTRACT

Industrialisation, directly or indirectly, exposes humans to various xenobiotics. The increased magnitude of chemical pesticides and toxic heavy metals in the environment, as well as their intrusion into the food chain, seriously threatens human health. Therefore, the surveillance of xenobiotics is crucial for social safety and security. Online investigation by traditional methods is not sufficient for the detection and identification of such compounds because of the high costs and their complexity. Advancement in the field of genetic engineering provides a potential opportunity to use genetically modified microorganisms. In this regard, whole-cell-based microbial biosensors (WCBMB) represent an essential tool that couples genetically engineered organisms with an operator/promoter derived from a heavy metal-resistant operon combined with a regulatory protein in the gene circuit. The plasmid controls the expression of the reporter gene, such as gfp, luc, lux and lacZ, to an inducible gene promoter and has been widely applied to assay toxicity and bioavailability. This review summarises the recent trends in the development and application of microbial biosensors and the use of mobile genes for biomedical and environmental safety concerns.


Subject(s)
Biosensing Techniques/methods , Environmental Monitoring/methods , Gene Expression Regulation , Organisms, Genetically Modified/metabolism , Synthetic Biology , Xenobiotics/analysis , Bacteria/genetics , Bacteria/metabolism , Genes, Reporter , Genetic Engineering , Hydrocarbons/toxicity , Metals, Heavy/toxicity , Microbial Sensitivity Tests , Pesticides/toxicity , Promoter Regions, Genetic , Yeasts/genetics , Yeasts/metabolism
4.
Aquat Toxicol ; 233: 105790, 2021 Apr.
Article in English | MEDLINE | ID: mdl-33662879

ABSTRACT

The potential exposure of titanate nanotubes (TNTs) to wildlife and humans may occur as a result of increased use and application as functional nanomaterials. However, there is a dearth of knowledge regarding the pathways of uptake and excretion of TNTs and their toxicity in cells. In this study, three strains of the Tetrahymena genus of free-living ciliates, including a wild type strain (SB210) and two mutant strains (SB255: mucocyst-deficient; NP1: temperature-sensitive "mouthless''), were used to study the pathways of uptake and excretion and evaluate the cytotoxicity of TNTs. The three Tetrahymena strains were separately exposed to 0, 0.01, 0.1, 1 or 10 mg/L of TNTs, and cells were collected at different time points for quantification of intracellular TNTs (e.g., 5, 10, 20, 40, 60, 90 and 120 min) and evaluation of cytotoxicity (12 and 24 h). TNT contents in NP1 and SB255 were greater or comparable to the contents in SB210 while exposure to 10 mg/L TNTs in 120 min. Furthermore, exposure to 10 mg/L TNTs for 24 h caused greater decreases in cell density of NP1 (38.2 %) and SB255 (36.8 %) compared with SB210 (26.5 %) and upregulated the expression of caspase 15 in SB210. Taken together, our results suggested that TNT uptake by pinocytosis and excretion by exocytosis in Tetrahymena, and the exposure could cause cytotoxicity which can offer novel insights into the accumulation kinetics of nanotubes and even nanomaterials in single cell.


Subject(s)
Nanotubes/toxicity , Organisms, Genetically Modified/drug effects , Tetrahymena/drug effects , Titanium/toxicity , Water Pollutants, Chemical/toxicity , Biological Transport , Coloring Agents , Dose-Response Relationship, Drug , Exocytosis/drug effects , Humans , Kinetics , Organisms, Genetically Modified/metabolism , Pinocytosis/drug effects , Tetrahymena/genetics , Tetrahymena/metabolism , Titanium/metabolism , Water Pollutants, Chemical/metabolism
5.
Carbohydr Polym ; 246: 116570, 2020 Oct 15.
Article in English | MEDLINE | ID: mdl-32747242

ABSTRACT

Chondroitin sulfate (CS) extracted from animal tissues has been widely used as nutraceutical and pharmaceutical products for osteoarthritis treatment. Here we developed an efficient sulfation-modification system for large scale preparation of CSA in vitro. First, the expression level of C4ST was improved by 30 times with fusion of the chaperone SUMO. Then, glycerol as a protein stabilizer was found to improve rat AST IV stability during the regeneration of cofactor PAPS. Then peptide linkers or protein scaffolds were employed to assemble AST IV and C4ST into artificial complexes to bring the enzymes and PAPS spatially closer and enhance the catalytic efficiency of chondroitin sulfation. Eventually, the system was scaled up to 1 L system and 15 g chondroitin was converted to CSA in 24 h, with a 98 % conversion. The present study made a step further towards the industrial production of CSA with different sulfation degrees.


Subject(s)
Arylsulfotransferase/metabolism , Chondroitin Sulfates/biosynthesis , Metabolic Engineering/methods , Sulfotransferases/metabolism , Adenosine Diphosphate/metabolism , Animals , Escherichia coli/enzymology , Escherichia coli/genetics , Kinetics , Organisms, Genetically Modified/metabolism , Plasmids/genetics , Rats , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomycetales/genetics , Saccharomycetales/metabolism , Solubility , Synthetic Biology/methods
6.
Sci Rep ; 10(1): 12713, 2020 07 29.
Article in English | MEDLINE | ID: mdl-32728100

ABSTRACT

Chlorella is a unicellular green microalga that has been used in fields such as bioenergy production and food supplementation. In this study, two promoters of N (nitrogen) deficiency-inducible Chlorella vulgaris N Deficiency Inducible (CvNDI) genes were isolated from Chlorella vulgaris UTEX 395. These promoters were used for the production of a recombinant protein, human granulocyte-colony stimulating factor (hG-CSF) in Chlorella vulgaris UTEX 395 and Chlorella sp. ArM0029B. To efficiently secrete the hG-CSF, the protein expression vectors incorporated novel signal peptides obtained from a secretomics analysis of Chlorella spp. After a stable transformation of those vectors with a codon-optimized hG-CSF sequence, hG-CSF polypeptides were successfully produced in the spent media of the transgenic Chlorella. To our knowledge, this is the first report of recombinant protein expression using endogenous gene components of Chlorella.


Subject(s)
Chlorella vulgaris/growth & development , Granulocyte Colony-Stimulating Factor/metabolism , Nitrogen/metabolism , Promoter Regions, Genetic , Algal Proteins/genetics , Algal Proteins/metabolism , Chlorella vulgaris/genetics , Chlorella vulgaris/metabolism , Granulocyte Colony-Stimulating Factor/genetics , Humans , Organisms, Genetically Modified/growth & development , Organisms, Genetically Modified/metabolism , Protein Engineering , Recombinant Proteins/metabolism
7.
J Biosci Bioeng ; 130(2): 149-158, 2020 Aug.
Article in English | MEDLINE | ID: mdl-32414665

ABSTRACT

The hyperthermophilic archaeon Thermococcus kodakarensis can grow on pyruvate or maltooligosaccharides through H2 fermentation. H2 production levels of members of the Thermococcales are high, and studies to improve their production potential have been reported. Although H2 production is primary metabolism, here we aimed to partially uncouple cell growth and H2 production of T. kodakarensis. Additional A1-type ATPase genes were introduced into T. kodakarensis KU216 under the control of two promoters; the strong constitutive cell surface glycoprotein promoter, Pcsg, and the sugar-inducible fructose-1,6-bisphosphate aldolase promoter, Pfba. Whereas cells with the A1-type ATPase genes under the control of Pcsg displayed only trace levels of growth, cells with Pfba (strain KUA-PF) displayed growth sufficient for further analysis. Increased levels of A1-type ATPase protein were detected in KUA-PF cells grown on pyruvate or maltodextrin, when compared to the levels in the host strain KU216. The growth and H2 production levels of strain KUA-PF with pyruvate or maltodextrin as a carbon and electron source were analyzed and compared to those of the host strain KU216. Compared to a small decrease in total H2 production, significantly larger decreases in cell growth were observed, resulting in an increase in cell-specific H2 production. Quantification of the substrate also revealed that ATPase overexpression led to increased cell-specific pyruvate and maltodextrin consumptions. The results clearly indicate that ATPase production results in partial uncoupling of cell growth and H2 production in T. kodakarensis.


Subject(s)
Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Gene Expression Regulation, Archaeal , Hydrogen/metabolism , Thermococcus/enzymology , Thermococcus/genetics , Carbon/metabolism , Gene Dosage/physiology , Gene Expression Regulation, Archaeal/genetics , Organisms, Genetically Modified/metabolism , Polysaccharides/metabolism , Pyruvic Acid/metabolism
8.
Microbiol Res ; 236: 126455, 2020 Jun.
Article in English | MEDLINE | ID: mdl-32179389

ABSTRACT

Echinenone and canthaxanthin are important carotenoid pigments with food and industrial applications. Biosynthesis of echinenone and/or canthaxanthin is catalyzed by ß-carotene ketolase (CrtO), with ß-carotene as the substrate. In this study, we generated transgenic Nostoc sp. PCC 7120 overexpressing a heterologous crtO gene from Nostoc flagelliforme and evaluated the productivity of both pigments. Normal (BG11 medium, 30 °C) and osmotic stress (BG11 medium supplemented with 0.4 M mannitol, 30 °C) conditions were used for cultivation. As compared to control strain, production of echinenone and canthaxanthin in transgenic strain were respectively increased by more than 16 % and 80 %, under either normal or osmotic stress conditions. Especially upon the stress condition, higher proportion of echinenone and canthaxanthin in total pigments was achieved, which should be beneficial for downstream separation and purification. In addition, transgenic strain showed drought tolerance and could revive from desiccation treatment after rewetting. Thus, this study provided technical clues for production of both pigments in engineered cyanobacteria as well as for cyanobacterial anhydrobiotic engineering.


Subject(s)
Nostoc/genetics , Organisms, Genetically Modified/growth & development , Organisms, Genetically Modified/metabolism , Oxygenases/genetics , Adaptation, Physiological , Bacterial Proteins/genetics , Canthaxanthin/biosynthesis , Carotenoids/metabolism , Cloning, Molecular , Droughts , Genes, Bacterial , Metabolic Engineering/methods , Nostoc/growth & development , Nostoc/metabolism , Organisms, Genetically Modified/genetics , Oxygenases/metabolism , beta Carotene/biosynthesis
9.
FEMS Microbiol Lett ; 367(6)2020 03 01.
Article in English | MEDLINE | ID: mdl-32129852

ABSTRACT

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.


Subject(s)
Fatty Acids/biosynthesis , Oils/metabolism , Organisms, Genetically Modified/metabolism , Waste Disposal, Fluid/methods , Yarrowia/metabolism , Erucic Acids/metabolism , Fatty Acid Desaturases/genetics , Genes, Plant/genetics , Organisms, Genetically Modified/genetics , Thlaspi/genetics , Yarrowia/genetics
10.
Microb Cell Fact ; 18(1): 194, 2019 Nov 07.
Article in English | MEDLINE | ID: mdl-31699086

ABSTRACT

BACKGROUND: 5'-Aminolevulinic acid (ALA) is widely used in the pharmaceutical industry, healthcare, and food production, and is a substrate for the biosynthesis of heme, which is required for respiration and photosynthesis. Enhancement of ALA biosynthesis has never been developed in Saccharomyces cerevisiae, which is a well-known model microorganism used for bioproduction of many value-added compounds. RESULTS: We demonstrated that metabolic engineering significantly improved ALA production in S. cerevisiae. First, we found that overexpression of HEM1, which encodes ALA synthetase, increased ALA production. Furthermore, addition of an optimal amount of glycine, a substrate for ALA biosynthesis, or levulinic acid, an inhibitor of ALA dehydrogenase, effectively increased ALA production. Next, we developed an assay for multiple metabolites including ALA and found that aconitase, encoded by ACO1 and ACO2, is the rate-limiting enzyme of ALA biosynthesis when sufficient glycine is supplied. Overexpression of ACO2 further enhanced ALA production in S. cerevisiae overexpressing HEM1. CONCLUSIONS: In this study, ALA production in S. cerevisiae was enhanced by metabolic engineering. This study also shows a strategy to identify the rate-limiting step of a target synthetic pathway by assay for multiple metabolites alongside the target product. This strategy can be applied to improve production of other valuable products in the well-studied and well-industrialized microorganism S. cerevisiae.


Subject(s)
Levulinic Acids/metabolism , Metabolic Engineering/methods , Organisms, Genetically Modified/metabolism , Saccharomyces cerevisiae , Aconitate Hydratase/genetics , Aconitate Hydratase/metabolism , Fermentation , Glycine/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Aminolevulinic Acid
11.
Microb Cell Fact ; 18(1): 195, 2019 Nov 07.
Article in English | MEDLINE | ID: mdl-31699116

ABSTRACT

BACKGROUND: The biological synthesis of high value compounds in industry through metabolically engineered microorganism factories has received increasing attention in recent years. Valencene is a high value ingredient in the flavor and fragrance industry, but the low concentration in nature and high cost of extraction limits its application. Saccharomyces cerevisiae, generally recognized as safe, is one of the most commonly used gene expression hosts. Construction of S. cerevisiae cell factory to achieve high production of valencene will be attractive. RESULTS: Valencene was successfully biosynthesized after introducing valencene synthase into S. cerevisiae BJ5464. A significant increase in valencene yield was observed after down-regulation or knock-out of squalene synthesis and other inhibiting factors (such as erg9, rox1) in mevalonate (MVA) pathway using a recyclable CRISPR/Cas9 system constructed in this study through the introduction of Cre/loxP. To increase the supplement of the precursor farnesyl pyrophosphate (FPP), all the genes of FPP upstream in MVA pathway were overexpressed in yeast genome. Furthermore, valencene expression cassettes containing different promoters and terminators were compared, and PHXT7-VS-TTPI1 was found to have excellent performance in valencene production. Finally, after fed-batch fermentation in 3 L bioreactor, valencene production titer reached 539.3 mg/L with about 160-fold improvement compared to the initial titer, which is the highest reported valencene yield. CONCLUSIONS: This study achieved high production of valencene in S. cerevisiae through metabolic engineering and optimization of expression cassette, providing good example of microbial overproduction of valuable chemical products. The construction of recyclable plasmid was useful for multiple gene editing as well.


Subject(s)
Metabolic Engineering/methods , Organisms, Genetically Modified/metabolism , Saccharomyces cerevisiae , Sesquiterpenes/metabolism , CRISPR-Cas Systems/genetics , Plasmids/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
12.
Microb Cell Fact ; 18(1): 178, 2019 Oct 21.
Article in English | MEDLINE | ID: mdl-31638987

ABSTRACT

The use of fossil fuels has been strongly related to critical problems currently affecting society, such as: global warming, global greenhouse effects and pollution. These problems have affected the homeostasis of living organisms worldwide at an alarming rate. Due to this, it is imperative to look for alternatives to the use of fossil fuels and one of the relevant substitutes are biofuels. There are different types of biofuels (categories and generations) that have been previously explored, but recently, the use of microalgae has been strongly considered for the production of biofuels since they present a series of advantages over other biofuel production sources: (a) they don't need arable land to grow and therefore do not compete with food crops (like biofuels produced from corn, sugar cane and other plants) and; (b) they exhibit rapid biomass production containing high oil contents, at least 15 to 20 times higher than land based oleaginous crops. Hence, these unicellular photosynthetic microorganisms have received great attention from researches to use them in the large-scale production of biofuels. However, one disadvantage of using microalgae is the high economic cost due to the low-yields of lipid content in the microalgae biomass. Thus, development of different methods to enhance microalgae biomass, as well as lipid content in the microalgae cells, would lead to the development of a sustainable low-cost process to produce biofuels. Within the last 10 years, many studies have reported different methods and strategies to induce lipid production to obtain higher lipid accumulation in the biomass of microalgae cells; however, there is not a comprehensive review in the literature that highlights, compares and discusses these strategies. Here, we review these strategies which include modulating light intensity in cultures, controlling and varying CO2 levels and temperature, inducing nutrient starvation in the culture, the implementation of stress by incorporating heavy metal or inducing a high salinity condition, and the use of metabolic and genetic engineering techniques coupled with nanotechnology.


Subject(s)
Biofuels , Lipids/biosynthesis , Metabolic Engineering/methods , Microalgae , Fermentation , Microalgae/genetics , Microalgae/growth & development , Microalgae/metabolism , Organisms, Genetically Modified/genetics , Organisms, Genetically Modified/metabolism
13.
Microb Cell Fact ; 18(1): 82, 2019 May 14.
Article in English | MEDLINE | ID: mdl-31088458

ABSTRACT

Microorganisms have long been used as chemical plant to convert simple substrates into complex molecules. Various metabolic pathways have been optimised over the past few decades, but the progresses were limited due to our finite knowledge on metabolism. Evolution is a knowledge-free genetic randomisation approach, employed to improve the chemical production in microbial cell factories. However, evolution of large, complex pathway was a great challenge. The invention of continuous culturing systems and in vivo genetic diversification technologies have changed the way how laboratory evolution is conducted, render optimisation of large, complex pathway possible. In vivo genetic diversification, phenotypic selection, and continuous cultivation are the key elements in in vivo continuous evolution, where any human intervention in the process is prohibited. This approach is crucial in highly efficient evolution strategy of metabolic pathway evolution.


Subject(s)
Fermentation , Industrial Microbiology , Metabolic Engineering , Metabolic Networks and Pathways , Organisms, Genetically Modified/metabolism , Secondary Metabolism
14.
BMC Microbiol ; 19(1): 7, 2019 01 08.
Article in English | MEDLINE | ID: mdl-30621597

ABSTRACT

BACKGROUND: Vulvovaginal candidiasis (VVC) is a common vaginitis caused by Candida species,a frequently recurring condition. Fungal azole-resistant strains with azole-resistance have developed for long and wide explosion to the first-line antifungal azole agent. Bovine lactoferrin (BLF) is a protein from transferrin family secreted by the bovine mammary tissue. Its various biological functions are well known, especially the pronounced antifungal activity. RESULTS: In the current study, we constructed a Lactobacillus casei strain (L.casei/pPG612.1-BLF), which secreted BLF encoded by a mature secretion vector plasmid pPG612.1, and evaluated its antifungal activity in vitro and in vivo. In a two-layer agar plate in vitro assay, the number of C. albicans CFUs decreased and the average colony size shrunk upon exposure to L. casei/pPG612.1-BLF. In a murine VVC model, the infection burden of mice intra-vaginally pre-inoculated with L. casei/pPG612.1-BLF was lower than in control groups. Furthermore, the infection burden in mice with VVC was reduced when the animals were continually given L. casei/pPG612.1-BLF as a topical treatment for 5 days. CONCLUSION: Combined, these results suggested that the L. casei/pPG612.1-BLF strain is a promising preventative and therapeutic anti-VVC agent, highlighting the possibility of employing the probiotic L. casei as a vehicle for biotherapy in the female genital tract and exploiting the natural antibiotic antimicrobial peptides for other applications.


Subject(s)
Candida albicans/drug effects , Candidiasis, Vulvovaginal/microbiology , Lacticaseibacillus casei/physiology , Lactoferrin/pharmacology , Probiotics/pharmacology , Animals , Antifungal Agents/metabolism , Antifungal Agents/pharmacology , Antifungal Agents/therapeutic use , Candidiasis, Vulvovaginal/drug therapy , Disease Models, Animal , Female , Lactoferrin/genetics , Lactoferrin/metabolism , Mice , Organisms, Genetically Modified/chemistry , Organisms, Genetically Modified/genetics , Organisms, Genetically Modified/metabolism , Plasmids/genetics , Probiotics/metabolism
15.
Chem Biol Drug Des ; 93(3): 300-312, 2019 03.
Article in English | MEDLINE | ID: mdl-30320974

ABSTRACT

Malaria is a devastating disease depending only on chemotherapy as treatment. However, medication is losing efficacy, and therefore, there is an urgent need for the discovery of novel pharmaceutics. Recently, plasmepsin V, an aspartic protease anchored in the endoplasmaic reticulum, was demonstrated as responsible for the trafficking of parasite-derived proteins to the erythrocytic surface and further validated as a drug target. In this sense, ligand-based virtual screening has been applied to design inhibitors that target plasmepsin V of P. falciparum (PMV). After screening 5.5 million compounds, four novel plasmepsin inhibitors have been identified which were subsequently analyzed for the potency at the cellular level. Since PMV is membrane-anchored, the verification in vivo by using transgenic PMV overexpressing P. falciparum cells has been performed in order to evaluate drug efficacy. Two lead compounds, revealing IC50 values were 44.2 and 19.1 µm, have been identified targeting plasmepsin V in vivo and do not significantly affect the cell viability of human cells up to 300 µm. We herein report the use of the consensus of individual virtual screening as a new technique to design new ligands, and we propose two new lead compounds as novel protease inhibitors to target malaria.


Subject(s)
Antimalarials/chemistry , Aspartic Acid Endopeptidases/metabolism , Ligands , Plasmodium falciparum/metabolism , Protozoan Proteins/metabolism , Antimalarials/metabolism , Antimalarials/pharmacology , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/genetics , Binding Sites , Catalytic Domain , Cell Survival/drug effects , Hep G2 Cells , Humans , Inhibitory Concentration 50 , Molecular Docking Simulation , Organisms, Genetically Modified/metabolism , Plasmodium falciparum/drug effects , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/genetics
16.
Methods Mol Biol ; 1859: 209-224, 2019.
Article in English | MEDLINE | ID: mdl-30421231

ABSTRACT

Isoprenoids are a highly diverse group of natural products with broad application as high value chemicals and advanced biofuels. They are synthesized using two primary building blocks, namely, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) that are generated via the mevalonate (MVA) or deoxy-D-xylulose-5-phosphate (DXP) pathways. Isoprenoid biosynthetic pathways are prevalent in eukaryotes, archaea, and bacteria. Measurement of isoprenoid intermediates via standard liquid chromatography-mass spectrometry (LC-MS) protocols is generally challenging because of the hydrophilicity and complex physicochemical properties of the molecules. In addition, there is currently no reliable analytical method that can simultaneously measure metabolic intermediates from MVA and DXP pathways, including the prenyl diphosphates. Therefore, we describe a robust hydrophilic interaction liquid chromatography time-of-flight mass spectrometry (HILIC-TOF-MS) method for analyzing isoprenoid intermediates from metabolically engineered Escherichia coli strains.


Subject(s)
Escherichia coli/metabolism , Hemiterpenes/analysis , Mass Spectrometry/methods , Metabolomics/methods , Organophosphorus Compounds/analysis , Biosynthetic Pathways/genetics , Chromatography, Liquid/instrumentation , Chromatography, Liquid/methods , Escherichia coli/genetics , Hemiterpenes/metabolism , Hydrophobic and Hydrophilic Interactions , Mass Spectrometry/instrumentation , Metabolic Engineering/instrumentation , Metabolic Engineering/methods , Metabolomics/instrumentation , Organisms, Genetically Modified/genetics , Organisms, Genetically Modified/metabolism , Organophosphorus Compounds/metabolism
17.
FEMS Yeast Res ; 18(8)2018 12 01.
Article in English | MEDLINE | ID: mdl-30124827

ABSTRACT

We report the permanent introduction of the human peroxisomal ß-oxidation enzymatic machinery required for straight chain degradation of fatty acids into the yeast, Saccharomyces cerevisiae. Peroxisomal ß-oxidation encompasses four sequential reactions that are confined to three enzymes. The genes encoding human acyl-CoA oxidase 1, peroxisomal multifunctional enzyme type 2 and 3-ketoacyl-CoA thiolase were introduced into the genomic loci of their yeast gene equivalents. The human ß-oxidation genes were individually tagged with sequence coding for GFP and expression of the protein chimeras as well as their targeting to peroxisomes was confirmed. Functional complementation of the ß-oxidation pathway was assessed by growth on media containing fatty acids of different chain lengths. Yeast cells exhibited distinctive substrate specificities depending on whether they expressed the human or their endogenous ß-oxidation machinery. The genetic engineering of yeast to contain a 'humanized' organelle is a first step for the in vivo study of human peroxisome disorders in a model organism.


Subject(s)
Fatty Acids/metabolism , Peroxisomes/enzymology , Peroxisomes/metabolism , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/metabolism , Genetic Complementation Test , Humans , Organisms, Genetically Modified/enzymology , Organisms, Genetically Modified/genetics , Organisms, Genetically Modified/metabolism , Oxidation-Reduction , Peroxisomes/genetics , Recombinant Proteins/genetics , Saccharomyces cerevisiae/genetics
18.
Appl Microbiol Biotechnol ; 102(12): 5021-5031, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29705960

ABSTRACT

Microbial production of solvents like acetone and butanol was a couple of the first industrial fermentation processes to gain global importance. These solvents are important feedstocks for the chemical and biofuel industry. Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H2 and CO2 under aerobic conditions. This bacterium is a natural producer of polyhydroxyalkanoate biopolymers. Recently, with the advances in the development of genetic engineering tools, the range of metabolites R. eutropha can produce has enlarged. Its ability to utilize various carbon sources renders it an interesting candidate host for synthesis of renewable biofuel and solvent production. This review focuses on progress in metabolic engineering of R. eutropha for the production of alcohols, terpenes, methyl ketones, and alka(e)nes using various resources. Biological synthesis of solvents still presents the challenge of high production costs and competition from chemical synthesis. Better understanding of R. eutropha biology will support efforts to engineer and develop superior microbial strains for solvent production. Continued research on multiple fronts is required to engineer R. eutropha for truly sustainable and economical solvent production.


Subject(s)
Biofuels , Carbon/metabolism , Cupriavidus necator/genetics , Cupriavidus necator/metabolism , Industrial Microbiology/methods , Solvents , Metabolic Engineering , Organisms, Genetically Modified/genetics , Organisms, Genetically Modified/metabolism
19.
Appl Microbiol Biotechnol ; 102(13): 5775-5783, 2018 Jul.
Article in English | MEDLINE | ID: mdl-29691627

ABSTRACT

The conversion of solar energy into hydrogen represents a highly attractive strategy for the production of renewable energies. Photosynthetic microorganisms have the ability to produce H2 from sunlight but several obstacles must be overcome before obtaining a sustainable and efficient H2 production system. Cyanobacteria harbor [NiFe] hydrogenases required for the consumption of H2. As a result, their H2 production rates are low, which makes them not suitable for a high yield production. On the other hand, [FeFe] enzymes originating from anaerobic organisms such as Clostridium exhibit much higher H2 production activities, but their sensitivity to O2 inhibition impairs their use in photosynthetic organisms. To reach such a goal, it is therefore important to protect the hydrogenase from O2. The diazotrophic filamentous cyanobacteria protect their nitrogenases from O2 by differentiating micro-oxic cells called heterocysts. Producing [FeFe] hydrogenase in the heterocyst is an attractive strategy to take advantage of their potential in a photosynthetic microorganism. Here, we present a biological engineering approach for producing an active [FeFe] hydrogenase (HydA) from Clostridium acetobutylicum in the heterocysts of the filamentous cyanobacterium Nostoc PCC7120. To further decrease the O2 amount inside the heterocyst, the GlbN cyanoglobin from Nostoc commune was coproduced with HydA in the heterocyst. The engineered strain produced 400 µmol-H2 per mg Chlorophyll a, which represents 20-fold the amount produced by the wild type strain. This result is a clear demonstration that it is possible to associate oxygenic photosynthesis with H2 production by an O2-sensitive hydrogenase.


Subject(s)
Clostridium acetobutylicum/enzymology , Hydrogen/metabolism , Hydrogenase/genetics , Hydrogenase/metabolism , Industrial Microbiology/methods , Nostoc/genetics , Organisms, Genetically Modified/genetics , Organisms, Genetically Modified/metabolism
20.
GM Crops Food ; 9(2): 53-58, 2018.
Article in English | MEDLINE | ID: mdl-29561212

ABSTRACT

Biotech news coverage in English-language Russian media fits the profile of the Russian information warfare strategy described in recent military reports. This raises the question of whether Russia views the dissemination of anti-GMO information as just one of many divisive issues it can exploit as part of its information war, or if GMOs serve more expansive disruptive purposes. Distinctive patterns in Russian news provide evidence of a coordinated information campaign that could turn public opinion against genetic engineering. The recent branding of Russian agriculture as the ecologically clean alternative to genetically engineered foods is suggestive of an economic motive behind the information campaign against western biotechnologies.


Subject(s)
Organisms, Genetically Modified/metabolism , Agriculture , Russia , Social Media
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