JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Effects of 20 Standard Amino Acids on the Growth, Total Fatty Acids Production, and ?-Linolenic Acid Yield in Mucor circinelloides.
Curr. Microbiol.
PUBLISHED: 08-13-2014
Show Abstract
Hide Abstract
Twenty standard amino acids were examined as single nitrogen source on the growth, total fatty acids production, and yield of ?-linolenic acid (GLA) in Mucor circinelloides. Of the amino acids, tyrosine gave the highest biomass and lipid accumulation and thus resulted in a high GLA yield with respective values of 17.8 g/L, 23 % (w/w, dry cell weight, DCW), and 0.81 g/L, which were 36, 25, and 72 % higher than when the fungus was grown with ammonium tartrate. To find out the potential mechanism underlying the increased lipid accumulation of M. circinelloides when grown on tyrosine, the activity of lipogenic enzymes of the fungus during lipid accumulation phase was measured. The enzyme activities of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and ATP-citrate lyase were up-regulated, while NADP-isocitrate dehydrogenase was down-regulated by tyrosine during the lipid accumulation phase of the fungus which suggested that these enzymes may be involved in the increased lipid biosynthesis by tyrosine in this fungus.
Related JoVE Video
Increased fatty acid unsaturation and production of arachidonic acid by homologous over-expression of the mitochondrial malic enzyme in Mortierella alpina.
Biotechnol. Lett.
PUBLISHED: 03-06-2014
Show Abstract
Hide Abstract
Malic enzyme (ME) catalyses the oxidative decarboxylation of L-malate to pyruvate and provides NADPH for intracellular metabolism, such as fatty acid synthesis. Here, the mitochondrial ME (mME) gene from Mortierella alpina was homologously over-expressed. Compared with controls, fungal arachidonic acid (ARA; 20:4 n-6) content increased by 60 % without affecting the total fatty acid content. Our results suggest that enhancing mME activity may be an effective mean to increase industrial production of ARA in M. alpina.
Related JoVE Video
Role of malic enzyme during fatty acid synthesis in the oleaginous fungus Mortierella alpina.
Appl. Environ. Microbiol.
PUBLISHED: 02-14-2014
Show Abstract
Hide Abstract
The generation of NADPH by malic enzyme (ME) was postulated to be a rate-limiting step during fatty acid synthesis in oleaginous fungi, based primarily on the results from research focusing on ME in Mucor circinelloides. This hypothesis is challenged by a recent study showing that leucine metabolism, rather than ME, is critical for fatty acid synthesis in M. circinelloides. To clarify this, the gene encoding ME isoform E from Mortierella alpina was homologously expressed. ME overexpression increased the fatty acid content by 30% compared to that for a control. Our results suggest that ME may not be the sole rate-limiting enzyme, but does play a role, during fatty acid synthesis in oleaginous fungi.
Related JoVE Video
Expression, purification, and characterization of NADP+-dependent malic enzyme from the oleaginous fungus Mortierella alpina.
Appl. Biochem. Biotechnol.
PUBLISHED: 01-26-2014
Show Abstract
Hide Abstract
Malic enzymes are a class of oxidative decarboxylases that catalyze the oxidative decarboxylation of malate to pyruvate and carbon dioxide, with concomitant reduction of NAD(P)+ to NAD(P)H. The NADP+-dependent malic enzyme in oleaginous fungi plays a key role in fatty acid biosynthesis. In this study, the malic enzyme-encoding complementary DNA (cDNA) (malE1) from the oleaginous fungus Mortierella alpina was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant protein (MaME) was purified using Ni-NTA affinity chromatography. The purified enzyme used NADP+ as the cofactor. The K m values for L-malate and NADP+ were 2.19±0.01 and 0.38±0.02 mM, respectively, while the V max values were 147±2 and 302±14 U/mg, respectively, at the optimal condition of pH 7.5 and 33 °C. MaME is active in the presence of Mn2+, Mg2+, Co2+, Ni2+, and low concentrations of Zn2+ rather than Ca2+, Cu2+, or high concentrations of Zn2+. Oxaloacetic acid and glyoxylate inhibited the MaME activity by competing with malate, and their K i values were 0.08 and 0.6 mM, respectively.
Related JoVE Video
The Roles of Moonlighting Proteins in Bacteria.
Curr Issues Mol Biol
PUBLISHED: 07-23-2013
Show Abstract
Hide Abstract
Moonlighting proteins, characterized by their multiple autonomous functions, have been detected in bacteria. Surprisingly, many of these proteins are conserved and involved in metabolic pathway or the cell stress response. They localise to the bacterial surface to take on additional activities, which have been hypothesised to contribute to bacterial virulence or bacterial benefit. In this review, we compare the functions of moonlighting proteins in bacteria, describe the structural basis of moonlighting functions, and summarise the regulation of secretion and localisation of moonlighting proteins.
Related JoVE Video
Regulatory properties of malic enzyme in the oleaginous yeast, Yarrowia lipolytica, and its non-involvement in lipid accumulation.
Biotechnol. Lett.
PUBLISHED: 05-14-2013
Show Abstract
Hide Abstract
Malic enzyme (EC 1.1.1.40) converts L-malate to pyruvate and CO2 providing NADPH for metabolism especially for lipid biosynthesis in oleaginous microorganisms. However, its role in the oleaginous yeast, Yarrowia lipolytica, is unclear. We have cloned the malic enzyme gene (YALI0E18634g) from Y. lipolytica into pET28a, expressed it in Escherichia coli and purified the recombinant protein (YlME). YlME used NAD(+) as the primary cofactor. Km values for NAD(+) and NADP(+) were 0.63 and 3.9 mM, respectively. Citrate, isocitrate and ?-ketoglutaric acid (>5 mM) were inhibitory while succinate (5-15 mM) increased NADP(+)- but not NAD(+)-dependent activity. To determine if fatty acid biosynthesis could be increased in Y. lipolytica by providing additional NADPH from an NADP(+)-dependent malic enzyme, the malic enzyme gene (mce2) from an oleaginous fungus, Mortierella alpina, was expressed in Y. lipolytica. No significant changes occurred in lipid content or fatty acid profiles suggesting that malic enzyme is not the main source of NADPH for lipid accumulation in Y. lipolytica.
Related JoVE Video
Role of the phenylalanine-hydroxylating system in aromatic substance degradation and lipid metabolism in the oleaginous fungus Mortierella alpina.
Appl. Environ. Microbiol.
PUBLISHED: 03-15-2013
Show Abstract
Hide Abstract
Mortierella alpina is a filamentous fungus commonly found in soil that is able to produce lipids in the form of triacylglycerols that account for up to 50% of its dry weight. Analysis of the M. alpina genome suggests that there is a phenylalanine-hydroxylating system for the catabolism of phenylalanine, which has never been found in fungi before. We characterized the phenylalanine-hydroxylating system in M. alpina to explore its role in phenylalanine metabolism and its relationship to lipid biosynthesis. Significant changes were found in the profile of fatty acids in M. alpina grown on medium containing an inhibitor of the phenylalanine-hydroxylating system compared to M. alpina grown on medium without inhibitor. Genes encoding enzymes involved in the phenylalanine-hydroxylating system (phenylalanine hydroxylase [PAH], pterin-4?-carbinolamine dehydratase, and dihydropteridine reductase) were expressed heterologously in Escherichia coli, and the resulting proteins were purified to homogeneity. Their enzymatic activity was investigated by high-performance liquid chromatography (HPLC) or visible (Vis)-UV spectroscopy. Two functional PAH enzymes were observed, encoded by distinct gene copies. A novel role for tetrahydrobiopterin in fungi as a cofactor for PAH, which is similar to its function in higher life forms, is suggested. This study establishes a novel scheme for the fungal degradation of an aromatic substance (phenylalanine) and suggests that the phenylalanine-hydroxylating system is functionally significant in lipid metabolism.
Related JoVE Video
Genome-scale analysis of the metabolic networks of oleaginous Zygomycete fungi.
Gene
PUBLISHED: 03-07-2013
Show Abstract
Hide Abstract
Microbial lipids are becoming an attractive option for the industrial production of foods and oleochemicals. To investigate the lipid physiology of the oleaginous microorganisms, at the system level, genome-scale metabolic networks of Mortierella alpina and Mucor circinelloides were constructed using bioinformatics and systems biology. As scaffolds for integrated data analysis focusing on lipid production, consensus metabolic routes governing fatty acid synthesis, and lipid storage and mobilisation were identified by comparative analysis of developed metabolic networks. Unique metabolic features were identified in individual fungi, particularly in NADPH metabolism and sterol biosynthesis, which might be related to differences in fungal lipid phenotypes. The frameworks detailing the metabolic relationship between M. alpina and M. circinelloides generated in this study is useful for further elucidation of the microbial oleaginicity, which might lead to the production improvement of microbial oils as alternative feedstocks for oleochemical industry.
Related JoVE Video
How are the non-classically secreted bacterial proteins released into the extracellular milieu?
Curr. Microbiol.
PUBLISHED: 02-25-2013
Show Abstract
Hide Abstract
Most bacterial proteins that are destined to leave the cytoplasm are exported across the cell membrane to their sites of function. These proteins are generally exported via the classical secretion pathway, in which the signal peptide plays a central role. However, some bacterial proteins have been found in the extracellular milieu without any apparent signal peptide. As none of the classical secretion systems is involved in their secretion, this occurrence is termed non-classical protein secretion. The mechanism or mechanisms responsible for non-classical secretion are contentious. This review compiles evidence from the debate over whether the release of the non-classically secreted proteins is the result of cell lysis and discusses how these proteins are exported to the exterior of the cell.
Related JoVE Video
Genetic engineering of Yarrowia lipolytica for enhanced production of trans-10, cis-12 conjugated linoleic acid.
Microb. Cell Fact.
PUBLISHED: 02-05-2013
Show Abstract
Hide Abstract
Conjugated linoleic acid (CLA) has been extensively studied for decades because of its health benefits including cancer prevention, anti-atherogenic and anti-obesity effects, and modulation of the immune system. We previously described the production of trans-10, cis-12 CLA in Yarrowia lipolytica by expressing the gene coding for linoleic acid isomerase from Propionibacterium acnes (pai). However the stable strain produced CLA at about 0.08% of dry cell weight (DCW), a level of production which was not high enough for practical applications. The goal of the present study was to enhance production of CLA by genetic engineering of Y. lipolytica strains.
Related JoVE Video
Optimizing lactose hydrolysis by computer-guided modification of the catalytic site of a wild-type enzyme.
Mol. Divers.
PUBLISHED: 01-23-2013
Show Abstract
Hide Abstract
Lactose intolerance is a serious global health problem. A lactose hydrolysis enzyme, thermostable ?-galactosidase, BgaB (from Geobacillus stearothermophilus) has attracted the attention of industrial biologists because of its potential application in processing lactose-containing products. However, this enzyme experiences galactose product inhibition. Through homology modeling and molecular dynamics (MD) simulation, we have identified the galactose binding sites in the thermostable ?-galactosidase BgaB (BgaB). The binding sites are formed from Glu303, Asn310, Trp311, His354, Arg109, Phe341, Try272, Asn147, Glu148, and H354; these residues are all important for enzyme catalysis. A ligand-receptor binding model has been proposed to guide site-directed BgaB mutagenesis experiments. Based upon the model and the MD simulations, we recommend mutating Arg109, Phe341, Trp311, Asn147, Asn310, Try272, and His354 to reduce galactose product inhibition. In vitro site-directed mutagenesis experiments confirmed our predictions. The success rate for mutagenesis was 66.7 %. The best BgaB mutant, F341T, can hydrolyze lactose completely, and is the most promising enzyme for use by the dairy industry. Thus, our study is a successful example of optimizing enzyme catalytic chemical reaction by computer-guided modifying the catalytic site of a wild-type enzyme.
Related JoVE Video
The secretion of an intrinsically disordered protein with different secretion signals in Bacillus subtilis.
Curr. Microbiol.
PUBLISHED: 01-12-2013
Show Abstract
Hide Abstract
In this study, a naturally unsecretory intrinsically disordered domain of nucleoskeletal-like protein (Nsp) was attempted to be secreted with different types of secretion signals in Bacillus subtilis. The results showed that Nsp can be secreted efficiently by all selected Sec-type signal peptides. Nsp was successfully exported when fused to Tat-type signal peptides but less efficient than Sec-type. The fusion protein with the non-classical extracellular proteins can be detected in the cell and extracellular milieu. This study further demonstrated that the mature protein plays an important role in protein secretion. Moreover, these results indicated that Nsp could be a useful tool to understand the individual roles of mature proteins and signal peptide in protein secretion, to evaluate the effect of conformation of mature proteins on their export pathway when coupled with Tat-type signal peptide, and to seek the signal of non-classical secretory proteins.
Related JoVE Video
Genome characterization of the oleaginous fungus Mortierella alpina.
PLoS ONE
PUBLISHED: 09-06-2011
Show Abstract
Hide Abstract
Mortierella alpina is an oleaginous fungus which can produce lipids accounting for up to 50% of its dry weight in the form of triacylglycerols. It is used commercially for the production of arachidonic acid. Using a combination of high throughput sequencing and lipid profiling, we have assembled the M. alpina genome, mapped its lipogenesis pathway and determined its major lipid species. The 38.38 Mb M. alpina genome shows a high degree of gene duplications. Approximately 50% of its 12,796 gene models, and 60% of genes in the predicted lipogenesis pathway, belong to multigene families. Notably, M. alpina has 18 lipase genes, of which 11 contain the class 2 lipase domain and may share a similar function. M. alpinas fatty acid synthase is a single polypeptide containing all of the catalytic domains required for fatty acid synthesis from acetyl-CoA and malonyl-CoA, whereas in many fungi this enzyme is comprised of two polypeptides. Major lipids were profiled to confirm the products predicted in the lipogenesis pathway. M. alpina produces a complex mixture of glycerolipids, glycerophospholipids and sphingolipids. In contrast, only two major sterol lipids, desmosterol and 24(28)-methylene-cholesterol, were detected. Phylogenetic analysis based on genes involved in lipid metabolism suggests that oleaginous fungi may have acquired their lipogenic capacity during evolution after the divergence of Ascomycota, Basidiomycota, Chytridiomycota and Mucoromycota. Our study provides the first draft genome and comprehensive lipid profile for M. alpina, and lays the foundation for possible genetic engineering of M. alpina to produce higher levels and diverse contents of dietary lipids.
Related JoVE Video
Cloning, expression, and identification of a novel class IIa bacteriocin in the Escherichia coli cell-free protein expression system.
Biotechnol. Lett.
PUBLISHED: 07-24-2011
Show Abstract
Hide Abstract
The NB-C1 gene, acquired from the result of data mining of the lactic acid bacteria genome, is a novel potential class IIa bacteriocin gene with the characteristic YGNGVxC cluster. To produce soluble NB-C1 efficiently and overcome issues of protein toxicity, we adopted a GFP fusion strategy using an Escherichia coli cell-free protein expression system. We constructed the expression vector pIVEX2.4d-GFP-NB-C1, which was expressed in both the batch mode and the continuous exchange cell-free (CECF) systems. The amount of soluble fusion protein achieved from the CECF system (2.2 mg/ml) was approximately three times higher than that in the batch mode (0.73 mg/ml). The soluble fusion protein was purified via one-step Ni-NTA affinity chromatography, with a concentration of 0.26 mg/ml and a purity of 95%. The purified NB-C1 showed strong antimicrobial activity against the indicator bacteria Listeria monocytogenes.
Related JoVE Video
Autotaxin: a protein with two faces.
Biochem. Biophys. Res. Commun.
PUBLISHED: 09-20-2010
Show Abstract
Hide Abstract
Autotaxin (ATX) is a catalytic protein, which possesses lysophospholipase D activity, and thus involved in cellular membrane lipid metabolism and remodeling. Primarily, ATX was thought as a culprit protein for cancer, which potently stimulates cancer cell proliferation and tumor cell motility, augments the tumorigenicity and induces angiogenic responses. The product of ATX catalyzed reaction, lysophosphatidic acid (LPA) is a potent mitogen, which facilitates cell proliferation and migration, neurite retraction, platelet aggregation, smooth muscle contraction, actin stress formation and cytokine and chemokine secretion. In addition to LPA formation, later ATX has been found to catalyze the formation of cyclic phosphatidic acid (cPA), which have antitumor role by antimitogenic regulation of cell cycle, inhibition of cancer invasion and metastasis. Furthermore, the very attractive information to the scientists is that the LPA/cPA formation can be altered at different physiological conditions. Thus the dual role of ATX with the scope of product manipulation has made ATX a novel target for cancer treatment.
Related JoVE Video
Large scale purification and characterization of recombinant human autotaxin/lysophospholipase D from mammalian cells.
BMB Rep
PUBLISHED: 08-28-2010
Show Abstract
Hide Abstract
We utilized a mammalian expression system to purify and characterize autotaxin (ATX)/lysophospholipase D, an enzyme present in the blood responsible for biosynthesis of lysophosphatidic acid. The human ATX cDNA encoding amino acids 29-915 was cloned downstream of a secretion signal of CD5. At the carboxyl terminus was a thrombin cleavage site followed by the constant domain (Fc) of IgG to facilitate protein purification. The ATX-Fc fusion protein was expressed in HEK293 cells and isolated from conditioned medium of a stable clone by affinity chromatography with Protein A sepharose followed by cleavage with thrombin. The untagged ATX protein was further purified to essential homogeneity by gel filtration chromatography with a yield of approximately 5 mg/liter medium. The purified ATX protein was enzymatically active and biologically functional, offering a useful tool for further biological and structural studies of this important enzyme.
Related JoVE Video
Sp-1 and c-Myc mediate lysophosphatidic acid-induced expression of vascular endothelial growth factor in ovarian cancer cells via a hypoxia-inducible factor-1-independent mechanism.
Clin. Cancer Res.
PUBLISHED: 01-17-2009
Show Abstract
Hide Abstract
Lysophosphatidic acid (LPA), which is present in ascites of ovarian cancer patients, stimulates expression of vascular endothelial growth factor (VEGF). VEGF is essential for the development and abdominal dissemination of ovarian cancer. We examined how LPA drives VEGF expression to gain a better understanding of tumor angiogenesis under normoxic conditions.
Related JoVE Video
Myosin-cross-reactive antigens from four different lactic acid bacteria are fatty acid hydratases.
Biotechnol. Lett.
Show Abstract
Hide Abstract
The 67 kDa myosin-cross-reactive antigen (MCRA) is a member of the MCRA family of proteins present in a wide range of bacteria and was predicted to have fatty acid isomerase function. We have now characterised the catalytic activity of MCRAs from four LAB stains, including Lactobacillus rhamnosus LGG, L. plantarum ST-III, L. acidophilus NCFM and Bifidobacterium animalis subsp. lactis BB-12. MCRA genes from these strains were cloned and expressed in Escherichia coli, and the recombinant protein function was analysed with lipid profiles by GC-MS. The four MCRAs catalysed the conversion of linoleic acid and oleic acid to their respective 10-hydroxy derivatives, which suggests that MCRA proteins catalyse the first step in conjugated linoleic acid production. This is the first report of MCRA from L. rhamnosus with such catalytic function.
Related JoVE Video
De novo synthesis of trans-10, cis-12 conjugated linoleic acid in oleaginous yeast Yarrowia lipolytica.
Microb. Cell Fact.
Show Abstract
Hide Abstract
Conjugated linoleic acid (CLA) has many well-documented beneficial physiological effects. Due to the insufficient natural supply of CLA and low specificity of chemically produced CLA, an effective and isomer-specific production process is required for medicinal and nutritional purposes.
Related JoVE Video
Annotation and analysis of malic enzyme genes encoding for multiple isoforms in the fungus Mucor circinelloides CBS 277.49.
Biotechnol. Lett.
Show Abstract
Hide Abstract
Based on the newly-released genomic data of Mucor circinelloides CBS 277.49, we have annotated five genes encoding for malic enzyme: all code for proteins that contain conserved domains/motifs for malic acid binding, NAD(+) binding and NAD(P)(+) binding. Phylogenetic analysis for malic enzyme genes showed that genes ID 78524 and 11639 share ~80% amino acid identity and are grouped in cluster 1; genes ID 182779, 186772 and 116127 share ~66% amino acid identity are grouped in cluster 2. Genes ID 78524, 11639 and 166127 produce proteins that are localized in the mitochondrion, while the products from genes 182779 and 186772 are localized in the cytosol. Based on the comparative analysis published previously by Song et al. (Microbiology 147:1507-1515, 2001), we propose that malic enzyme genes ID 78524, 166127, 182779, 186772, 11639, respectively, represent protein isoforms I, II, III/IV, V, and VI.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.