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PubMed1997

D-lysine production from L-lysine by successive chemical racemization and microbial asymmetric degradation.

In order to develop a practical process for D-lysine production from L-lysine, successive chemical racemization and microbial asymmetric degradation were investigated. The racemization of L-lysine proceeded quantitatively at elevated temperatures. A sample of 1000 strains of bacteria, fungi, yeast and actinomyces were screened for the ability to degrade L-lysine asymmetrically. Microorganisms belonging to the Achromobacter, Agrobacterium, Candida, Comamonas, Flavobacterium, Proteus, Providencia, Pseudomonas and Yarrowia genera exhibited a high L-lysine-degrading activity. Comamonas testosteroni IAM 1048 was determined to be the best strain and used as a biocatalyst for eliminating the L isomer. The degradation rate of L-lysine with C. testosteroni IAM 1048 was influenced by pH, temperature and agitation speed. Under the optimal conditions, the L isomer in a 100-g/l mixture of racemic lysine was completely degraded within 72 h, with 47 g D-lysine/l left in the reaction mixture. Crystalline D-lysine, with a chemical purity greater than 99% and optical purity of 99.9% enantiomeric excess, was obtained at a yield of 38% from the reaction mixture by simple purification. An engineering analysis of L-lysine racemization and microbial degradation was carried out to establish the basis of process design for D-lysine production.

Biodegradation, Environmental↗
PubMed1985

Single-stranded hexameric linkers: a system for in-phase insertion mutagenesis and protein engineering.

An efficient method for introducing two (or four) codons into a cloned gene has been developed. Single-stranded (ss) hexameric linkers are inserted into a plasmid linearized at cohesive-end restriction sites. The resultant 6 (or 12)-bp insertion creates a new 6-bp restriction site. Plasmids containing linker insertions are enriched by using biochemical selection, or selected by using a kanamycin-resistance (KmR) cassette (biological selection). A total of 57 new linkers have been designed, and compatible KmR cassettes flanked by eleven different restriction sites have been constructed. Two-codon insertions into the tetracycline-resistance (TcR) gene of pBR322 yielded a series of new plasmid vectors. Moreover, proteins with internally duplicated domains have been constructed from beta-lactamase (ApR) insertions into the ApR gene of pBR322. Some of the resulting "gemini" proteins retained the beta-lactamase activity.

Cloning, Molecular↗
PubMed1988

Biodegradation of hydrocarbons in the environment.

Studies on the environmental fate of petroleum have demonstrated the nearly ubiquitous distribution of microorganisms that can metabolize hydrocarbons. The rates of degradation depend upon the concentrations of such microbes and upon the environmental characteristics of an oil-contaminated ecosystem. Given the appropriate environmental conditions, microorganisms effectively decontaminate, by their biodegradative metabolism, environments that have received petroleum pollutants. Higher-molecular-weight compounds, especially those with multiple condensed ring structures and with highly branched or substituted compounds, are relatively resistant to microbial attack. Despite the fact that a genetically engineered hydrocarbon degrader was the first organism ever patented and that seed cultures are produced by various commercial firms, enhanced biodegradation as a result of seeding generally has not been shown to be effective. Also, even though some anaerobes have now been demonstrated to be capable of hydrocarbon metabolism, hydrocarbons persist indefinitely in anoxic environments. Environmental modification, on the other hand, such as that achieved by aeration or fertilization with nitrogen and phosphorus, has been shown to enhance biodegradative removal of hydrocarbons. Having considered the various factors that influence the rates of hydrocarbon biodegradation, we are left with the question of what to do when environmental oil contamination occurs in order to minimize its persistence and thus its long-term effects. Clearly, treatment methods should enhance rather than inhibit the natural rates of oil biodegradation. In some cases, it is possible to modify environmental parameters to enhance rates of hydrocarbon biodegradation, but such methods are rarely undertaken. The translation of our scientific knowledge of hydrocarbon biodegradation into practical applications remains a major challenge. Specifically designed organisms are needed to degrade toxic aromatic components of refinery waste streams before environmental treatment. Specially designed reactors with specific microbial populations are also needed if oily sludges are to be degraded by biological means, either aerobically or anaerobically, in contained, environmentally safe reactors.

Biodegradation, Environmental↗
PubMed2006

Plant genetic engineering to improve biomass characteristics for biofuels.

Currently, most ethanol produced in the United States is derived from maize kernel, at levels in excess of four billion gallons per year. Plant lignocellulosic biomass is renewable, cheap and globally available at 10-50 billion tons per year. At present, plant biomass is converted to fermentable sugars for the production of biofuels using pretreatment processes that disrupt the lignocellulose and remove the lignin, thus allowing the access of microbial enzymes for cellulose deconstruction. Both the pretreatments and the production of enzymes in microbial tanks are expensive. Recent advances in plant genetic engineering could reduce biomass conversion costs by developing crop varieties with less lignin, crops that self-produce cellulase enzymes for cellulose degradation and ligninase enzymes for lignin degradation, or plants that have increased cellulose or an overall biomass yield.

Biomass↗
PubMed1994

Immunogenicity of microbial peptides grafted in self immunoglobulin molecules.

The advent of genetic engineering has allowed for the expression and production of recombinant proteins carrying short immunogenic epitopes of foreign antigens. These antigenized molecules represent valuable tools to investigate the molecular basis of antigen fragmentation, generation and presentation of peptide to lymphocytes, the induction of epitope specific immunity and potentially the development of a new generation of vaccines. Recently, we expressed viral epitopes on immunoglobulin molecules by replacing the D segment of a variable region of the heavy chain (VH) gene with a B cell epitope from the V3-loop of HIV-1 envelope protein, as well as a cytotoxic T lymphocyte (CTL) and a T helper epitope from influenza virus nucleoprotein and hemagglutinin, respectively. The T cell peptides generated from the immunoglobulin molecules produced by cells transfected with chimeric V genes, activated specific T cells as they do when generated from viral proteins. Possible practical applications for the development of prophylactic and immunotherapeutic reagents are envisioned for immunoglobulin molecules bearing foreign epitopes.

Amino Acid Sequence↗
PubMed1990

Survival of genetically engineered Escherichia coli in natural soil and river water.

Twelve derivatives of Escherichia coli strain HB101 which contained different sizes of plasmids ranging from 3.9 Kb to 48 Kb and encoding resistance to various antibiotics were used. When these organisms were introduced into natural river water, the population declined rapidly and by day 3, the majority (i.e. more than 99.9%) of them could no longer be detected on antibiotic-amended culture plates. If the river water was filter sterilized first, the added organisms maintained their population for up to 7 d without any significant decrease in numbers. Similar results were also observed in sterilized tap water or distilled water. This indicated that the disappearance of these organisms in the aquatic environment was caused mainly by biotic factor(s). The loss of the ability to grow in the presence of antibiotics by some of the E. coli was not observed unless they were allowed to grow in the antibiotic-free environment first. When the test organisms were added to natural silt loam, a large portion of the original population still remained viable after 16 d. There was no relationship between the percentage survival of E. coli in natural river water and the sizes of plasmid harboured. On the other hand, when these bacteria were added to natural soil, survival appeared to increase as plasmid size increased.

Drug Resistance, Microbial↗
PubMed2000

Bioluminescent most-probable-number monitoring of a genetically engineered bacterium during a long-term contained field release.

Pseudomonas fluorescens HK44 is a lux-based bioluminescent bioreporter capable of emitting light upon exposure to naphthalene, salicylate, and other substituted analogs. The bacterium was inoculated into intermediate-scale field lysimeters and population dynamics were monitored with time. Two methods were used to enumerate cell numbers in soil: a standard selective plating technique with colony hybridization verification and a modified lux-based most-probable-number (lux-MPN) assay based on the detection of bioluminescence. The lux-MPN assay was developed and evaluated as a possible supplement or replacement for the labor-intensive and time-consuming selective plating assay. Comparisons between selective plate counts and lux-MPN population estimates showed similar trends over the 2-year study, except that lux-MPN estimates were consistently less than selective plate counts. Verification of P. fluorescens HK44 genotype through colony hybridization techniques revealed that selective plating was actually overestimating HK44 populations and that lux-MPN values were more closely approximating true HK44 cell densities, except within the first few weeks after inoculation, when lux-MPN estimates underrepresented population densities. Thus, utilizing bioluminescence as a population monitoring tool for lux-based microorganisms was shown to be more effective and precise than standard selective plating techniques, and provided an accurate ecological analysis of P. fluorescens HK44 population dynamics over an extended period.

Colony Count, Microbial↗
PubMed1995

Recombinant and wild-type Pseudomonas aureofaciens strains introduced into soil microcosms: effect on decomposition of cellulose and straw.

The effect of a genetically engineered Pseudomonas aureofaciens (Ps3732RNL11) strain (GEM) and the parental wild-type (Ps3732RN) on decomposition of cellulose paper, straw and calico cloth was assessed after 18 weeks incubation in laboratory soil microcosms. Effect(s) of inoculum density (10(3), 10(5), and 10(8) cells/g dry soil) and single versus multiple bacterial inoculations were also investigated. Cellulose paper was completely decomposed after 18 weeks in all treatments. There were no significant differences (95% level), between treatments, in percentage decomposition of either straw or calico cloth. Recovery of the GEM at 18 weeks, using viable plating, was limited to treatments originally receiving 10(8) cells/g dry soil. Log 1.8 CFU/g dry soil were recovered from the single dose treatment while log 4.2 CFU/g dry soil were recovered from the multiple dose treatment. Biolog metabolic tests were used to determine if the GEM or parental wild-type had any effect on overall carbon utilization in soil. Results suggested they did not. Detection of the recombinant lacZY gene sequence in soil using PCR suggested the possibility of viable but nonculturable cells and/or persistence of chromosomal DNA.

Bacteria, Aerobic↗
PubMed1999

Colonization and clearance of environmental microbial agents upon intranasal exposure of strain C3H/HeJ mice.

Environmental dissemination of biotechnology agents is becoming a common practice. Most applications use historically innocuous species; however, potential health effects of individual products are not scrutinized unless they contain genetically engineered microorganisms. In order to investigate possible health concerns, four surrogate microbial agents were studied in vivo. Male C3H/HeJ (endotoxin-resistant) mice were administered intranasally (i.n.) with approximately 10(7) Pseudomonas aureofaciens, Burkholderia cepacia, P. fluorescens, or P. putida. To determine clearance of the dosed bacterial strains, lungs, small intestine, large intestine, cecum, mesenteric lymph nodes (MLN), spleen, and liver were homogenized individually, plated, and dilutions inoculated onto selective media. Pseudomonas fluorescens and P. putida were eliminated from the lungs by 2 d posttreatment, and P. aureofaciens was not detected in the lungs by 5 d posttreatment. Burkholderia cepacia was reisolated from the lungs and cecum for the experimental duration (14 d). Translocation to extraintestinal sites (MLN, spleen, and liver) also occurred. Burkholderia cepacia was recovered from the MLN for 10 d after treatment of mice. Pulmonary exposure to several bacterial strains resulted in unexpected mortality. Pseudomonas aureofaciens was lethal at the lowest dose (8.26 x 10(6) CFU/ mouse), while P. fluorescens and B. cepacia were fatal at higher doses (6.15 x 10(8) CFU/mouse and 1.34 x 10(8) CFU/mouse, respectively). By using the model described in this study, human safety issues can be more easily addressed and evaluated.

Administration, Intranasal↗
PubMed1999

Effect of organic acids on the growth and fermentation of ethanologenic Escherichia coli LY01.

Hemicellulose residues can be hydrolyzed into a sugar syrup using dilute mineral acids. Although this syrup represents a potential feedstock for biofuel production, toxic compounds generated during hydrolysis limit microbial metabolism. Escherichia coli LY01, an ethanologenic biocatalyst engineered to ferment the mixed sugars in hemicellulose syrups, has been tested for resistance to selected organic acids that are present in hemicellulose hydrolysates. Compounds tested include aromatic acids derived from lignin (ferulic, gallic, 4-hydroxybenzoic, syringic, and vanillic acids), acetic acid from the hydrolysis of acetylxylan, and others derived from sugar destruction (furoic, formic, levulinic, and caproic acids). Toxicity was related to hydrophobicity. Combinations of acids were roughly additive as inhibitors of cell growth. When tested at concentrations that inhibited growth by 80%, none appeared to strongly inhibit glycolysis and energy generation, or to disrupt membrane integrity. Toxicity was not markedly affected by inoculum size or incubation temperature. The toxicity of all acids except gallic acid was reduced by an increase in initial pH (from pH 6.0 to pH 7.0 to pH 8.0). Together, these results are consistent with the hypothesis that both aliphatic and mononuclear organic acids inhibit growth and ethanol production in LY01 by collapsing ion gradients and increasing internal anion concentrations.

Acids↗
PubMed2004

Recent progress in microbial cultivation techniques.

Recent advances in the improvement of microbial cultivation are reviewed, with emphasis on biochemical engineering techniques as a means of obtaining high production rate of bioproduct. Possible uses of high cell density culture include their use in food industry as well as in the production of new medicines and in biotechnology. Concentration of microorganisms using a hollow fiber membrane or centrifuge, and increase in cell density by controlling the pH, dissolved oxygen, or carbon source concentrations of the culture broth with control algorithms are discussed. In a culture of filamentous microorganisms the mycelial morphology is hard to define and it is difficult to quantify its amount, and this is one of the bottlenecks hampering the improvement of production rate. Specific features of mycelial cultivation in the presence of highly pulpy mycelia and entangled-pellets are scrutinized by visual inspection through a microscope that is linked to a computer, and using software that can characterize the mycelial morphology. Image analysis technology for analyzing the mycelial image captured by a digital camera is a potential tool for morphological analysis, including analysis of the morphological development of filamentous microorganisms.

Algorithms↗
PubMed2007

Construction of plasmid vector for expression of bacteriocin N15-encoding gene and effect of engineered bacteria on Enterococcus faecalis.

A 6.09-kb plasmids vector pOri253 was constructed from the plasmid pIL253 (5.2 kb) and a 0.89-kb fragment of oriColE1 from pBluescript II KS. The bifunctional plasmid pOri253 conferred erythromycin resistance in both Escherichia coli and Enterococcus faecalis. It has unique sites for EcoRI, BamHI, SalI, and PstI derived from pIL253 and was lost at a low rate in E. faecalis JCM8726 when cultured in Man, Rogosa, & Sharpe broth without antibiotic. The lactococcal promoter P23 was inserted at one end of the pOri253 multicloning site. Gene expression was assessed by an entAI gene, which produced bacteriocin N15. The E. faecalis harboring constructed plasmid carrying P23 (pOrient23) had more antibacterial activity than parental E. faecalis JCM8726 and its clone harboring non-P23-containing plasmid (pOrient), as determined by means of an overlay method.

Bacteriocins↗
PubMed1983

Stable integration of foreign DNA into the chromosome of the cyanobacterium Synechococcus R2.

The blue-green alga, Synechococcus R2, is transformed to antibiotic resistance by chimeric DNA molecules consisting of Synechococcus R2 chromosomal DNA linked to antibiotic-resistance genes from Escherichia coli. Chimeric DNA integrates into the Synechococcus R2 chromosome by homologous recombination. The efficiency of transformation, as well as the stability of integrated foreign DNA, depends on the position of the foreign genes relative to Synechococcus R2 DNA in the chimeric molecule. When the Synechococcus R2 DNA fragment is interrupted by foreign DNA, integration occurs through replacement of chromosomal DNA by homologous chimeric DNA containing the foreign insert; transformation is efficient and the foreign gene is stable. Mutagenesis in some cases attends integration, depending on the site of insertion. Foreign DNA linked to the ends of Synechococcus R2 DNA in a circular molecule, however, integrates less efficiently. Integration results in duplicate copies of Synechococcus R2 DNA flanking the foreign gene and the foreign DNA is unstable. Transformation in Synechococcus R2 can be exploited to modify precisely and extensively the genome of this photosynthetic microorganism.

Cyanobacteria↗
PubMed1986

Human adenovirus cloning vectors based on infectious bacterial plasmids.

By making use of the fact that human adenovirus DNA circularizes in infected cells, and that circular forms of the viral genome are infectious, we have developed an improved adenovirus-based cloning system. A deletion mutant of adenovirus type 5 (Ad5) with deletions in early regions 1 (E1) and 3 (E3) was converted to a bacterial plasmid which can regenerate infectious virus following transfection into human 293 cells. A single XbaI recognition site in the deleted E3 region serves as a site for the insertion of foreign DNA. We have used this system to clone a number of genes into the Ad5 genome and describe the insertion of the neomycin/G418 resistance marker into Ad5 as an example.

Adenoviruses, Human↗
PubMed2006

Suicide vectors for antibiotic marker exchange and rapid generation of multiple knockout mutants by allelic exchange in Gram-negative bacteria.

Allelic exchange is frequently used in bacteria to generate knockout mutants in genes of interest, to carry out phenotypic analysis and learn about their function. Frequently, understanding of gene function in complex processes such as pathogenesis requires the generation of multiple mutant strains. In Pseudomonads and other non-Enterobacteriaceae, this is a time-consuming and laborious process based on the use of suicide vectors and allelic exchange of the appropriate mutant version of each gene, disrupted by a different antibiotic marker. This often implies the generation of a series of mutants for each gene, each disrupted by a different antibiotic marker, in order to obtain all possible double or multiple mutant combinations. In this work, we have modified this method by developing a set of 3 plasmid derivatives from the previously described suicide vector for allelic exchange, pKAS32, to make antibiotic marker exchange easier and thus accelerate the entire process. Briefly, the construction of each single gene knockout mutant is carried out by allelic exchange of the chromosomal gene with a mutant allele disrupted by the insertion of a kanamycin resistance cassette. When a double mutant strain is required, antibiotic marker exchange is performed in either one of the single mutants, using any of the three plasmid derivatives that carry the kanamycin resistance gene disrupted by either a chloramphenicol, gentamycin, or streptomycin resistance cassette. The single mutant strain, carrying now an antibiotic resistance marker other than kanamycin, can be used to introduce a second mutation using the original plasmid constructs, to generate a double mutant. The process can be repeated sequentially to generate multiple mutants. We have validated this method by generating strains carrying different combinations of mutations in genes encoding different transcriptional regulators of the Hrp type III secretion system in Pseudomonas syringae. We have also tested the genetic organisation and stability of the resulting mutant strains during growth in laboratory conditions as well as in planta.

Anti-Bacterial Agents↗
PubMed2002

Display of proteins on bacteria.

Display of heterologous proteins on the surface of microorganisms, enabled by means of recombinant DNA technology, has become an increasingly used strategy in various applications in microbiology, biotechnology and vaccinology. Gram-negative, Gram-positive bacteria, viruses and phages are all being investigated in such applications. This review will focus on the bacterial display systems and applications. Live bacterial vaccine delivery vehicles are being developed through the surface display of foreign antigens on the bacterial surfaces. In this field, 'second generation' vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals, through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other type of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. Certain bacteria have also been employed for display of various poly-peptide libraries for use as devices in in vitro selection applications. Through various selection principles, individual clones with desired properties can be selected from such libraries. This article explains the basic principles of the different bacterial display systems, and discusses current uses and possible future trends of these emerging technologies.

Bacterial Proteins↗