Search

Retroviral vectors integrate in genes and regulatory elements and may cause transcriptional deregulation of gene expression in target cells. Integration into transcribed genes also has the potential to deregulate gene expression at the posttranscriptional level by interfering with splicing and polyadenylation of primary transcripts. To examine the impact of retroviral vector integration on transcript splicing, we transduced primary human cells or cultured cells with HIV-derived vectors carrying a reporter gene or a human [beta]-globin gene under the control of a reduced-size locus-control region (LCR). Cells were randomly cloned and integration sites were determined in individual clones. We identified aberrantly spliced, chimeric transcripts in more than half of the targeted genes in all cell types. Chimeric transcripts were generated through the use of constitutive and cryptic splice sites in the HIV 5' long terminal repeat and gag gene as well as in the [beta]-globin gene and LCR. Compared with constitutively spliced transcripts, most aberrant transcripts accumulated at a low level, at least in part as a consequence of nonsense-mediated mRNA degradation. A limited set of cryptic splice sites caused the majority of aberrant splicing events, providing a strategy for recoding lentiviral vector backbones and transgenes to reduce their potential posttranscriptional genotoxicity., Introduction Large-scale surveys of retroviral integration in murine and human cells uncovered genomic features systematically associated with retroviral insertions and revealed that each retrovirus type has a unique, characteristic pattern [...]

One of the limitations facing the development of Streptococcus gordonii into a successful vaccine vector is the inability of this bacterium to express high levels of heterologous proteins. In the present study, we have identified 12 codons deemed as rare codons in S. gordonii and seven other streptococcal species, tRNA genes encoding 10 of the 12 rare codons were cloned into a plasmid. The plasmid was transformed into strains of S. gordonii expressing the fusion protein SpaP/S1, the anti-complement receptor 1 (CR1) single-chain variable fragment (scFv) antibody, or the Toxoplasma gondii cyclophilin C18 protein. These three heterologous proteins contained high percentages of amino acids encoded by rare codons. The results showed that the production of SpaP/S1, anti-CR1 scFv and C18 increased by 2.7-, 120-and 10-fold, respectively, over the control strains. In contrast, the production of the streptococcal SpaP protein without the pertussis toxin S1 fragment was not affected by tRNA gene supplementation, indicating that the increased production of SpaP/S1 protein was due to the ability to overcome the limitation caused by rare codons required for the S1 fragment. The increase in anti-CR1 scFv production was also observed in Streptococcus mutans following tRNA gene supplementation. Collectively, the findings in the present study demonstrate for the first time, to the best of our knowledge, that codon-usage bias exists in Streptococcus spp. and the limitation of heterologous protein expression caused by codon-usage bias can be overcome by tRNA supplementation. DOI 10.1099/mic.0.030064-0

Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp NCIMB 9871 expressed in baker's yeast and in E. coil and cyclopentanone monooxygenase (CPMO) from Comamonas (previously Pseudomonas) sp. NCIMB 9872 expressed in E. coli are new bioreagents for Baeyer-Villiger oxidations. These engineered microorganisms, requiring neither biochemical expertise nor equipment beyond that found in chemical laboratories, were evaluated as reagents for Baeyer-Villiger oxidations of cyclopentanones substituted at the 2-position with polar and nonpolar chains suitable for further modifications. Two such functionalized substrates that can be transformed into highly enantiopure lactones were identified. The performance and the potential of these bioreagents are discussed. Key words: enantioselective Baeyer-Villiger oxidations, biotransformations, cyclohexanone monooxygenase, cyclopentanone monooxygenase, engineered baker's yeast, recombinant E. coli, optically pure 2-substituted cyclopentanones, optically pure lactones.

Researchers have bioengineered a strain of Rhizobium leguminosarum bv. viciae that can produce hydrogenase in free-living cells to the same extent that it would in UPM791 bacteroids. This strain, designated SPF25, was created by replacing the native NifA-dependent hupSL promoter with the FnrN-dependent fixN promoter.

Researchers used PCR-mediated random mutagenesis of the gene for polyhydroxyalkanoate (PHA) synthase in Aeromonas caviae to generate two versions of the enzyme that exhibited 21% and 56% increased activity toward 3-hydroxybutyrate-coenzyme A. This caused a 6.5-fold increase in the production of a PHA copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate.

The degree to which teichoic acids in the cell wall of Bacillus subtilis are alanylated determines the secretion of recombinant protective antigen of Bacillus anthracis. Protective antigen is used to create the anthrax vaccine so researchers are interested in creating a recombinant form in Bacillus subtilis. This will eliminate trace amounts of other proteins that can cause an adverse reaction to the vaccine.

The biodegradation of cellulose can be enhanced by genetically modifying Clostridium cellulolyticum to prevent the buildup of pyruvate, which inhibits its growth. This was done by inserting the genes for pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis. Cellulose consumption increased 150% and growth increased 180% compared to the wild strain.

Researchers discovered nine plasmids that can be introduced into Lactobacillus delbrueckii using electroporation. The plasmids were able to replicate and integrated an integrative vector. This bacterium is used as a starter in the dairy industry and may be a useful probiotic.

Researchers have created a bioengineered form of cytochrome P450 BM-3 from Bacillus megaterium that has higher activity against polycyclic aromatic hydrocarbons than the native enzyme. The mutant enzyme could be used to degrade these compounds in polluted soil.

Saccharomyces cerevisiae can create ethanol from xylose if it contains the xylA gene from Thermus thermophilus and the GRE3 yeast gene is removed. Xylose is a common sugar in lignocellulosic material, which is fermented to produce ethanol as an alternative to fossil fuels.

Researchers successfully bioengineered Saccharomyces cerevisiae with the beta-xylosidase gene of Aspergillus niger 90196 and the beta-xylanase gene from Trichoderma reesei. The coproduction of these two fungal enzymes allowing the yeast to degrade xylan. Beta-xylanases have industrial applications in the food and biobleaching industries.

Researchers describe a plasmid that can be used to insert any gene into the genome of Brevibacterium lactofermentum. This bacterium is used industrially to produce amino acids. The plasmid was used to insert the xysA gene from Streptomyces halstedii, which was stable for more than 200 generations.

Genetically engineered bacteria with synthetic phytochelatins (ECs) on their cell surface could be used to clean up toxic metals in the environment. Phytochelatins are naturally occurring metal-binding peptides and the synthetic version has the repetitive metal-binding motif (Glu-Cys)(sub n)Gly.

Saccharomyces cerevisiae can be genetically modified to produce medium-chain polyhydroxyalkanoate (PHA). PHA is of interest because it is a biodegradable polyester. The genetic modification includes the transformation of S. cerevisiae with the PHAC1 synthase from Pseudomonas aeruginosa targeted to the peroxisome.

A recombinant form of the nuclear polyhedrosis virus Autographa californica that does not produce an occlusion body in Trichoplusia ni insect larva can persist over several successive rounds of infection and has a multiplicity of infection of between 4 and 5 virus genomes per cell. The recombinant virus was co-occluded with the wild-type virus and used to infect fifth instar larvae.

Researchers show that only a small proportion of recombinant nucleopolyhedrovirus is transported by rain and wind to areas outside the treatment area. This virus could be used as a biological control agent for insects but it must be genetically engineered to increase its toxicity.

Researchers have genetically engineered Clostridium beijeinckii with glycoside hydrolase genes from the rumen fungus Neocallimastix patriciarum that allow C. beijerinckii to grow on lichenan. Clostridium spp. can produce butanol and ethanol from carbohydrates and biomass is the most widely available substrate for this conversion.

Researchers created a version of Staphylococcus carnosus that binds nickel by introducing a genetically engineered fungal cellulose-binding domain (CBD) from Trichoderma reesei cellulase. The CBD do longer bound cellulose but was able to bind metals. Metal-binding bacteria could be used in biofilters or biosensors.

Introducing an extra Cheo box into the promoter region of the tumor necrosis factor alpha gene in genetically engineered clostridia can increase the secretion of the protein when the bacterium is exposed to radiation. This system could be used to treat cancer patients because the treatment could be stopped at any time by killing the bacterium with antibiotics.

The gacS-cysM intergenic region of Pseudomonas syringae pv. syringae B728a can be used for site-directed insertion of markers to distinguish it from other bacteria in field experiments. Up to seven kb of DNA can be inserted without significantly altering bacterial fitness.

The introduction of a foreign gene into a microorganism can change the expression of native proteins. In a study of Saccharomyces cerevisiae and Lactococcus lactis, protein expression was altered between three- and 14-fold when the foreign reporter gene gusA was inserted.

Pseudomonas putida that has been genetically modified to produce the antifungal compound phenazine-1-carboxylic acid can suppress fungal growth in soil rhizospheres. The Antifungal compound is produced by the phz gene from Pseudomonas fluorescens 2-79.

Researchers have discovered a genetic sequence on the lactococcal plasmid pMRC01 that resembles an origin of transfer (oriT). This oriT could be used to in a vector to genetically modify lactococcal starter strains used in the food industry.

Researchers have created a recombinant form of tissue plasminogen activator that can be produced by E. coli with no requirement for bacterial compartment preparation or in vitro refolding. The protein is secreted into the culture supernatant and has a fibrin binding site and amidolytic activity.

Researchers describe a biological containment system for recombinant strains of Pseudomonas putida that are used in bioremediation. These systems contain a killing gene, usually a porin-coding gene, that is activated when the pollutant is eliminated. This destroys the recombinant bacteria, making it less likely that they will remain in the environment.

Researchers used genetic engineering to suppress the production of non-clavulanic acid clavams by Streptomyces clavuligerus. This bacterium is used to produce clavulanic acid, an antibiotic that can inhibit beta lactamases. It is used clinically in combination with beta lactam antibiotics.

Researchers have engineered a strain of yeast that displays a-agglutinin and alpha-agglutinin on the cell surface. This yeast grows normally at high glucose concentrations, but agglutinates and sediments completely when glucose concentrations are very low. This can be used industrially to separate the cells from the supernatant.

A replicon from the lactococcal plasmid pCD4 was used to introduce a mechanism for phage resistance into Lactococcus lactis strain MG1363 and two other industrial strains. The phenotype lasted over 100 generations and provided resistance to phage species 936 and c2.

Researchers have genetically engineered Lactococcus lactis to produce rotavirus nonstructural protein 4 (NSP4). NSP4 is believed to be an enterotoxin produced by rotavirus, which is a major cause of diarrhea in babies and children.

Researchers describe genetically engineering Saccaromyces cerevisiae with tadpole ferritin. Ferritin is a large macromolecule that can store up to 4,500 iron atoms. This genetically engineered form of yeast could be used as an iron supplement in cattle.

Laccase from the white-rot fungus Trametes versicolor can be expressed in Saccharomyces cerevisiae, which will make the yeast resistant to phenolic compounds produced during the fermentation of lignocellulose. Lignocellulose can be fermented to ethanol, which can be used as a fuel.

Genetically engineered viruses used for insect control may have different effects on insects that are susceptible to the virus and those that are not. This was the conclusion of researchers who studied the effect of a genetically modified baculovirus and its wild counterpart against Mamestra brassicae and Trichoplusia ni.

An electroporation-based transformation procedure to create an efficient host-vector system in Desulfitobacterium dehalogenans is described. This microorganism can degrade pollutants in soils, aquifers, and sediments.

Researchers used a 3.6-kb plasmid from Propionibacterium freudenreichii to insert an E.coli-specific replicon and an erythromycin resistance gene into the bacterium. This bacterium is used for industrial purposes, so a means of genetically modifying the bacterium would be useful.

Researchers describe a system to control multiple genes from a single promoter. This will allow scientists to engineer bacteria to produce multi-subunit proteins.

Researchers created a strain of E. coli that can accumulate nickel by introducing the nixA gene from Helicobacter pylori. This gene codes for a nickel transport system. The resulting strain accumulated four times as much nickel as the native strain.

Researchers describe a simple method for producing antibody fragments in E. coli using the E. coli hemolysin transport system. This system can produce the antibody fragment without a periplasmic intermediate stage.

Researchers were able to increase the secretion of an enzyme by Saccharomyces cerevisiae five-fold by putting an N-glycosylation consensus sequence in the N-terminal region. This technique could be used to enhance the preferential secretion of a particular protein for industrial purposes.

The lactose operon of Lactobacillus casei could be exploited to genetically engineer the bacterium to express genes for industrial, clinical, and veterinary uses. Researchers have used the operon to express the E. coli gusA gene and the L. lactis ilvBN gene in L. casei.

Advances in the genetic engineering of Thermoanaerobacterium spp. are discussed. This organism can survive at temperatures up to 80 deg. C. and can use xylan, which is a cheap substrate for industrial fermentation.

Researchers have created a mutant form of the enzyme tyrosine phenol-lyase from Erwinia herbicola that does not require L-tyrosine. This bacterium is used to produce L-DOPA for the treatment of Parkinson's disease, but the wild enzyme requires tyrosine. The tyrosine must then be separated from L-DOPA.

Researchers describe a plasmid that could be used to introduce various genes into Propionibacterium acidipropionici. This organism is used in the food and pharmaceutical industries.

Researcher demonstrate that genetically engineered bacteria that normally colonize the roots of poplar trees can survive in the rhizosphere and degrade trichloroethylene. Trichloroethylene is a common groundwater pollutant at hazardous waste sites. Poplar trees grow quickly and a single five-year-old tree could process 53 gallons of polluted water per day.

Researchers isolated the H3 and H4 histone genes from Mortierella alpina and used the H4 promoter in a transformation vector to induce the expression of a gene for hygromycin B resistance. This fungus is of interest to indusrial microbiologists because it produces long-chain polyunsaturated fatty acids.

Researchers used genetic engineering to create a strain of Saccharomyces cerevisiae that can produce ethanol from xylose. The native bacterium can only use xylulose, which is an isomer of xylose.

Researchers describe an autocloning method that produces multiple copies of the gene for extracellular lipase in Yarrowia lipolytica. These strains could produce 40 times more lipase per liter than strains containing only one copy of the gene. This makes them a promising source of lipase for industrial use, waste treatment, and pancreatic insufficiency treatment.