synthetic biology; genetic engineering; catabolic engineering; biodegradation; robustness; xenobiotics
Resistance development against multiple drugs is a common feature among many pathogens—including bacteria such as Pseudomonas aeruginosa, viruses, and parasites—and also among cancers. The reasons are two-fold. Most commonly-used rationally-designed small molecule drugs or monoclonal antibodies, as well as antibiotics, strongly inhibit a key single step in the growth and proliferation of the pathogen or cancer cells. The disease agents quickly change or switch off this single target, or activate the efflux mechanisms to pump out the drug, thereby becoming resistant to the drug. A second problem is the way drugs are designed. The pharmaceutical industry chooses to use, by high-throughput screening, compounds that are maximally inhibitory to the key single step in the growth of the pathogen or cancer, thereby promoting selective pressure. An ideal drug would be one that inhibits multiple steps in the disease progression pathways with less stringency in these steps. Low levels of inhibition at multiple steps provide cumulative strong inhibitory effect, but little incentives or ability on the part of the pathogen/cancer to develop resistance. Such intelligent drug design involving multiple less stringent inhibitory steps is beyond the scope of the drug industry and requires evolutionary wisdom commonly possessed by bacteria. This review surveys assessments of the current clinical situation with regard to drug resistance in P. aeruginosa, and examines tools currently employed to limit this trend. We then provide a conceptual framework in which we explore the similarities between multi-drug resistance in pathogens and in cancers. We summarize promising work on anti-cancer drugs derived from the evolutionary wisdom of bacteria such as P. aeruginosa, and how such strategies can be the basis for how to look for candidate protein/peptide antibiotic drugs from bioengineered bugs. Such multi-domain proteins, unlike diffusible antibiotics, are not diffusible because of their large size and are often released only on contact with the perceived competitor. Thus, multi-domain proteins are missed during traditional methods of looking for growth zone inhibition of susceptible bacteria as demonstrated by antibiotics, but may represent the weapons of the future in the fights against both drug-resistant cancers and pathogens such as P. aeruginosa.
Pseudomonas aeruginosa; anti-pseudomonal drugs; multi-drug resistance; azurin; new-generation protein/peptide drugs
Baculovirus is extensively utilized as an excellent tool for production of recombinant protein in insect cells. Baculovirus infects insects in nature and is non-pathogenic to humans. In addition to insect cells, baculovirus is capable of transducing a broad range of animal cells. Due to its biosafety, large cloning capacity, low cytotoxicity, and non-replication nature in the transduced cells as well as the ease of manipulation and production, baculovirus has been utilized as RNA interference mediators, gene delivery vectors, and vaccine vectors for a wide variety of applications. This article focuses on the utilization of baculoviruses as vaccine vectors to prepare antigen or subunit vaccines.
baculovirus; surface display vector; vaccine; VSVG; gp64
The properties of bacteriorhodopsin (BR) can be manipulated by genetic engineering. Therefore, by the methods of gene engineering, Asp85 was replaced individually by two other amino acids (D85V, D85S). The resulting recombinant proteins were assembled into soybean vesicles retinylated to form functional BR-like nano-particles. Proton translocation was almost completely abrogated by the mutant D85S, while the D85V mutant was partially active in pumping protons. Compared with wild type, maximum absorption of the mutants, D85V and D85S, were 563 and 609 nm, which illustrated 5 nm reductions (blue shift) and 41 nm increases (red shift), respectively. Since proton transport activity and spectroscopic activities of the mutants are different, a wide variety of membrane bioreactors (MBr) have been developed. Modified proteins can be utilized to produce unique photo/Electro-chromic materials and tools.
Absorption spectra; Bacteriorhodopsin; Mutants; Proton pumping; Site-directed mutagenesis
N-acetylglucosamine (GlcNAc), the monomer of chitin and constituent of bacterial peptidoglycan, is a preferred carbon and nitrogen source for streptomycetes. Recent studies have revealed new functions of GlcNAc in nutrient signaling of bacteria. Exposure to GlcNAc activates development and antibiotic production of Streptomyces coelicolor under poor growth conditions (famine) and blocks these processes under rich conditions (feast). Glucosamine-6-phosphate (GlcN-6P) is a key molecule in this signaling pathway and acts as an allosteric effector of a pleiotropic transcriptional repressor DasR, the regulon of which includes the GlcNAc metabolic enzymes N-actetylglucosamine-6-phosphate (GlcNAc-6P) deacetylase (NagA) and GlcN-6P deaminase (NagB). Intracellular accumulation of GlcNAc-6P and GlcN-6P enhanced production of the pigmented antibiotic actinorhodin. When the nagB mutant was challenged with GlcNAc or GlcN, spontaneous second-site mutations that relieved the toxicity of the accumulated sugar phosphates were obtained. Surprisingly, deletion of nagA also relieved toxicity of GlcN, indicating novel linkage between the GlcN and GlcNAc utilization pathways. The strongly enhanced antibiotic production observed for many suppressor mutants shows the potential of the modulation of GlcNAc and GlcN metabolism as a metabolic engineering tool toward the improvement of antibiotic productivity or even the discovery of novel compounds.
metabolism; aminosugar; signaling; glycolysis; secondary metabolism; sporulation
Affinity precipitation is a non-chromatographic method which is useful for purification and refolding of proteins. Quite often, a stimuli-sensitive polymer can be identified which selectively binds to the desired protein. For separation, the protein can be recovered from the precipitate of the protein-smart polymer complex. In case of a refolding experiment, binding of the solubilized protein (in its denatured form) with the polymer leads to the refolding of the protein.
smart polymer; bioseparation of proteins; protein refolding; affinity precipitation; protein purification; inclusion bodies; stimuli-sensitive polymers; water soluble polymers
Protein production through dedicated secretion systems might offer an potential alternative to the conventional cytoplasmical expression. The application of Type 1 secretion systems of Gram-negative bacteria, however, where often not successful in the past for a wide range of proteins. Recently, two studies using the E. coli maltose binding protein (MalE) and the rat intestinal fatty acid binding protein (IFABP) revealed a rational to circumvent these limitations. Here, wild-type passenger proteins were not secreted, while folding mutants with decreased folding kinetics were efficiently exported to the extracellular space. Subsequently, an one-step purification protocol yielded homogeneous and active protein. Taken together, theses two studies suggest that the introduction of slow-folding mutations into a protein sequence might be the key to use Type 1 secretion systems for the biotechnological production of proteins.
HlyB; IFABP; Type 1 secretion; maltose; mutation; protein folding; relaxation; western blot
Brucellosis is an important zoonotic disease of almost worldwide distribution. One significant immune phenomenon of this disease is the ability of the pathogen to hide and survive in the host, establishing long lasting chronic infections. Brucella was found to have the ability to actively modulate the host immune response in order to establish chronic infections, but the mechanism by which the pathogen achieves this remains largely unknown. In our screening for protective antigens of Brucella abortus, 3 proteins (BAB1_0597, BAB1_0917, and BAB2_0431) were found to induce significantly higher levels of gamma interferon (IFNγ) in splenocytes of PBS immunized mice than those immunized with S19. This finding strongly implied that these three proteins inhibit the production of IFNγ. Previous studies have shown that LPS, PrpA, and Btp1/TcpB are three important immunomodulatory molecules with the capacity to interfere with host immune response. They have been shown to have the ability to inhibit the secretion of IFNγ, or to increase the production of IL-10. Due to the role of these proteins in virulence and immunomodulation, they likely offer significant potential as live, attenuated Brucella vaccine candidates. Understanding the mechanisms by which these proteins modulate the host immune responses will deepen our knowledge of Brucella virulence and provide important information on the development of new vaccines against Brucellosis.
Brucella; chronic infection; immune response; live attenuated vaccine; virulence proteins
Increased stress tolerance of economically important plants and microorganisms can
improve yields in agriculture and industrial microbiology. The pool of resources used for
the genetic modification of crops and industrial fungal strains in the past has been
relatively limited, and has frequently included only stress-sensitive organisms. However,
certain groups of fungi have evolved specialized mechanisms that enable them to thrive
under even the most extreme of environmental conditions. These species can be considered
as promising sources of biotechnologically interesting genes. Together with a powerful and
convenient high-throughput functional screening method, extremotolerant fungi represent a
new opportunity for the identification of stress-tolerance-conferring genes. The
approaches described here should provide important contributions to the enhancing of the
properties of economically important organisms in the future.
extremotolerant organism; functional screening; gain-of-function method; genetically modified organism; halotolerance; salinisation; stress tolerance genes; stress-tolerant fungi
It has been documented that bacteria from the Burkholderia genera produce different kinds of compounds that inhibit plant pathogens, however in Burkholderia tropica, an endophytic diazotrophic and phosphate-solubilizing bacterium isolated from a wide diversity of plants, the capacity to produce antifungal compounds has not been evaluated. In order to expand our knowledge about Burkholderia tropica as a potential biological control agent, we analyzed 15 different strains of this bacterium to evaluate their capacities to inhibit the growth of four phytopathogenic fungi, Colletotrichum gloeosporioides, Fusarium culmorum, Fusarium oxysporum and Sclerotium rolffsi. Diverse analytical techniques, including plant root protection and dish plate growth assays and gas chromatography-mass spectroscopy showed that the fungal growth inhibition was intimately associated with the volatile compounds produced by B. tropica and, in particular, two bacterial strains (MTo293 and TTe203) exhibited the highest radial mycelial growth inhibition. Morphological changes associated with these compounds, such as disruption of fungal hyphae, were identified by using photomicrographic analysis. By using gas chromatography-mass spectroscopy technique, 18 volatile compounds involved in the growth inhibition mechanism were identified, including α-pinene and limonene. In addition, we found a high proportion of bacterial strains that produced siderophores during growth with different carbon sources, such as alanine and glutamic acid; however, their roles in the antagonism mechanism remain unclear.
Burkholderia tropica; volatile compound; antagonism; phytopathogenic fungi
Designing appropriate expression vectors is one of the critical steps in the generation of stable cell lines for recombinant protein production. Conventional expression vectors are severely affected by the chromatin environment surrounding their integration site into the host genome, resulting in low expression levels and transgene silencing. In the past, a new generation of expression vectors and different strategies was developed to overcome the chromatin effects. Bacterial artificial chromosomes (BACs) are cloning vectors capable of accommodating up to 350 Kb. Thus, BACs can carry a whole eukaryotic locus with all the elements controlling the expression of a gene; therefore, BACs harbor their own chromatin environment. Expression vectors based on BACs containing open/permissive chromatin loci are not affected by the chromatin surrounding their integration site in the host cell genome. Consequently, BAC-based expression vectors containing the appropriate loci confer predictable and high levels of expression over time. These properties make BAC-based expression vectors a very attractive tool applied to the recombinant protein production field.
bacterial artificial chromosome; chromatin; CHO cells; mammalian; expression vector; producer cell line; transgene; recombinant protein production
Genetic bioaugmentation is an in situ bioremediation method that stimulates horizontal transfer of catabolic plasmids between exogenous donor cells and indigenous bacteria to increase the biodegradation potential of contaminants. A critical outcome of genetic bioaugmentation is the expression of an active catabolic phenotype upon plasmid conjugation. Using a pWW0-derivative TOL plasmid, we showed that certain genetic characteristics of the recipient bacteria, including genomic guanine-cytosine (G + C) content and phylogeny, may limit the expression of the transferred catabolic pathway. However, such genetic limitations observed in transconjugants could be overcome by the presence of an additional carbon source. Glucose and Luria-Bertani broth were shown to enhance the toluene degradation rates of transconjugants; these enhancement effects were dependent on transconjugant genomic G + C contents. Based on these observations, thorough genetic characterization of the indigenous microbial community in the contaminated environment of interest may provide a predictive tool for assessing the success of genetic bioaugmentation.
TOL plasmid; bioremediation; carbon amendment; genetic bioaugmentation; guanine-cytosine content; plasmid conjugation
The heterologous expression of laccases is important for their large-scale production and genetic engineering—a prerequisite for industrial application. Pichia pastoris is the preferred expression host for fungal laccases. The recently cloned laccase from the ascomycete Botrytis aclada (BaLac) has been efficiently expressed in P. pastoris under the control of the inducible alcohol oxidase (AOX1) promoter. In this study, we compare these results to the constitutive expression in the same organism using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The results show that the amounts of BaLac produced with the GAP system (517 mgL-1) and the AOX1 system (495 mgL-1) are comparable. The constitutive expression is, however, faster, and the specific activity of BaLac in the culture supernatant is higher (41.3 Umg-1 GAP, 14.2 Umg-1 AOX1). In microtiter plates, the constitutive expression provides a clear advantage due to easy manipulation (simple medium, no methanol feeding) and fast enzyme production (high-throughput screening assays can already be performed after 48 h).
Botrytis aclada; Pichia pastoris; 96-well plate expression; GAP promoter; ascomycete; chloride tolerance; high yield; laccase screening; laccase; constitutive expression
The ability to generate tailor-made, functionalized polyester (polyhydroxyalkanoate, PHA) beads in bacteria by harnessing their natural carbon-storage granule production system is an exciting recent development. Proteins that naturally attach to the polyester granule core were rationally engineered to enable in vivo production of PHA beads which are applicable in bioseparation, protein purification, enzyme immobilization and diagnostics and which show advantageous properties toward the development of safe and efficient particulate vaccines. These beads are recombinantly produced as fully functional, insoluble polyester inclusions that can be easily separated from the cell. This simple one-step production of functionalized beads provides a tantalizing alternative to current commercial functional beads, for which proteins must be expressed, purified and then chemically attached to solid supports. The recent success in generating antigen-displaying PHA granules in the food-grade bacterium Lactococcus lactis capable of mediating protective immunity against Mycobacterium tuberculosis infection highlights the promise and flexibility of this new technology.
PHA synthase; PHA; PHB; bacterial inclusions; beads; biopolymer; bioseparation; polyester; polyhydroxyalkanoate; vaccines
Vaccination is the most cost effective strategy for the control and prevention of the plethora of viral diseases affecting poultry production. The major challenge for poultry vaccination is the design of vaccines that will protect against multiple pathogens via a single protective dose, delivered by mass vaccination. The Marek disease virus and the highly pathogenic avian influenza virus cause severe disease outbreaks in chickens. Vaccination with live herpesvirus of turkeys protects chickens from Marek disease and inactivated influenza viruses are used as antigens to protect chickens against influenza virus infections. We developed herpesvirus of turkeys (HVT) as a vaccine vector that can act as a dual vaccine against avian influenza and Marek disease. The HVT vector was developed using reverse genetics based on an infectious bacterial artificial chromosome (BAC) clone of HVT. The BAC carrying the HVT genome was genetically modified to express the haemagglutinin (HA) gene of a highly pathogenic H7N1 virus. The resultant recombinant BAC construct containing the modified HVT sequence was transfected into chicken embryo fibroblast (CEF) cells and HVT recombinants (rHVT-H7HA) harbouring the H7N1 HA were recovered. Analysis of cultured CEF cells infected with the rHVT-H7HA showed that HA was expressed and that the rescued rHVT-H7HA stocks were stable during several in vitro passages with no difference in growth kinetics compared with the parent HVT. Immunization of one-day-old chicks with rHVT-H7HA induced H7-specific antibodies and protected chickens challenged with homologous H7N1 virus against virus shedding, clinical disease and death. The rHVT-H7HA vaccine also induced strong and long-lasting antibody titers against H7HA in chickens that were vaccinated in ovo 3 d before hatching. This vaccine supports differentiation between infected and vaccinated animals (DIVA), because no influenza virus nucleoprotein-specific antibodies were detected in the rHVT-H7HA vaccinated birds. The rHVT-H7HA not only provided protection against a lethal challenge with highly pathogenic H7N1 virus but also against highly virulent Marek disease virus and can be used as a DIVA vaccine.
HVT BAC; Marek’s disease; avian; avian influenza; highly pathogenic H7N1 avian influenza virus; in ovo vaccination; multivalent vaccine; poultry; recombinant herpesvirus of turkeys; vector-based vaccine
Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Since Chinese hamster ovary (CHO) cells are capable of producing heparan sulfate (HS), a related polysaccharide naturally, and heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. We developed stable human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) expressing cell lines based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells. Both activity assay and disaccharide analysis showed that engineered HS attained heparin-like characteristics but not identical to pharmaceutical heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary.
Chinese hamster ovary cells; LC-MS; anticoagulant; heparin; metabolic engineering; transcriptional regulation; translational regulation
Metabolic engineering is often facilitated by cloning of genes encoding enzymes from various heterologous organisms into E. coli. Such engineering efforts are frequently hampered by foreign genes that are toxic to the E. coli host. We have developed PanDaTox (www.weizmann.ac.il/pandatox), a web-based resource that provides experimental toxicity information for more than 1.5 million genes from hundreds of different microbial genomes. The toxicity predictions, which were extensively experimentally verified, are based on serial cloning of genes into E. coli as part of the Sanger whole genome shotgun sequencing process. PanDaTox can accelerate metabolic engineering projects by allowing researchers to exclude toxic genes from the engineering plan and verify the clonability of selected genes before the actual metabolic engineering experiments are conducted.
gene cloning; metabolic engineering; pandatox; synthetic biology; toxic genes
Lignocellulosic biomass, upon pretreatment and enzymatic hydrolysis, generates a mixture of hexose and pentose sugars such as glucose, xylose, arabinose and galactose. While Escherichia coli utilizes all these sugars it lacks the ability to produce ethanol from them. Recombinant ethanologenic E. coli strains have been created with a goal to produce ethanol from both hexose and pentose sugars. Herein, we review the current state of the art on the production of ethanol from lignocellulosic hydrolyzates by an ethanologenic recombinant E. coli strain (FBR5). The bacterium is stable without antibiotics and can tolerate ethanol up to 50 gL-1. It produces up to 45 g ethanol per L and has the potential to be used for industrial production of ethanol from lignocellulosic hydrolyzates.
ethanol fermentation; lignocellulosic biomass; recombinant ethanologenic Escherichia coli; separate hydrolysis and fermentation; simultaneous saccharification and fermentation
Chagas disease is the clinical manifestation of the infection produced by the parasite Trypanosoma cruzi. Currently there is no vaccine to prevent this disease and the protection attained with vaccines containing non-replicating parasites is limited. Genetically attenuated trypanosomatid parasites can be obtained by deletion of selected genes. Gene deletion takes advantage of the fact that this parasite can undergo homologous recombination between endogenous and foreign DNA sequences artificially introduced in the cells. This approach facilitated the discovery of several unknown gene functions, as well as allowing us to speculate about the potential for genetically attenuated live organisms as experimental immunogens. Vaccination with live attenuated parasites has been used effectively in mice to reduce parasitemia and histological damage, and in dogs, to prevent vector-delivered infection in the field. However, the use of live parasites as immunogens is controversial due to the risk of reversion to a virulent phenotype. Herein, we present our results from experiments on genetic manipulation of two T. cruzi strains to produce parasites with impaired replication and infectivity, and using the mutation of the dhfr-ts gene as a safety device against reversion to virulence.
Trypanosoma cruzi; dhfr-ts; Chagas disease; gene knockout
Illicium verum (badiane or star anise), Crataegus oxyacantha ssp monogyna (hawthorn) and Allium cepa (onion), have traditionnally been used as medicinal plants in Algeria. This study showed that the outer layer of onion is rich in flavonols with contents of 103 ± 7.90 µg/g DW (red variety) and 17.3 ± 0.69 µg/gDW (white variety). We also determined flavonols contents of 14.3 ± 0.21 µg/g 1.65 ± 0.61 µg/g for Crataegus oxyacantha ssp monogyna leaves and berries and 2.37 ± 0.10 µg/g for Illicium verum. Quantitative analysis of anthocyanins showed highest content in Crataegus oxyacantha ssp monogyna berries (5.11 ± 0.266 mg/g), while, inner and outer layers of white onion had the lowest contents with 0.045 ± 0.003mg/g and 0.077 ± 0.001 mg/g respectively.
Flavonols extracts presented high antioxidant activity as compared with anthocyanins and standards antioxidants (ascorbic acid and quercetin). Allium cepa and Crataegus oxyacantha ssp monogyna exhibited the most effective antimicrobial activity.
flavonoids; flavonols; anthocyanins; antioxydant activity; antibacterial activity
With world wide data predicted to exceed 40 trillion gigabytes by 2020, big data storage is a very real and escalating problem. Herein, we discuss the utility of synthetic DNA as a robust and eco-friendly archival data storage solution of the future.