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PubMed2005

New aminocoumarin antibiotics from genetically engineered Streptomyces strains.

The aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A(1) are produced by different Streptomyces strains and are potent inhibitors of DNA gyrase. The biosynthetic gene clusters of all three antibiotics have been cloned and sequenced, and the function of most genes contained therein has been elucidated. In the last years, a number of "unnatural" aminocoumarins could be generated using the genetic information for the biosynthesis of these antibiotics. The investigated enzymes of aminocoumarin biosynthesis have less-than-perfect substrate specificity, facilitating the production of new antibiotics by various methods. Several new aminocoumarins could be produced by targeted genetic manipulation in the natural producers, but also in heterologous host Streptomyces coelicolor after expression of the respective gene cluster. Mutasynthesis experiments, i. e. generation of a cloQ-defective mutant of the clorobiocin producer and feeding of 13 different structural analogs of 3-dimethylallyl-4-hydroxybenzoic acid to this mutant, allowed the isolation of 32 new aminocoumarins. These compounds contained, instead of the genuine 3-dimethylallyl-4-hydroxybenzoyl moiety, the externally added analogs as the acyl components in their structures. Production of new aminocoumarins was also achieved by chemoenzymatic synthesis in vitro. Several biosynthetic enzymes have been heterologously expressed, purified und used for chemoenzymatic synthesis. The structures of the new aminocoumarins were elucidated by NMR and mass spectroscopy. Their inhibitory activity on gyrase in vitro as well as their antibacterial activity was determined. These results give further insight into the structure-activity relationships of aminocoumarins.

Animals↗
PubMed2006

Theoretical analysis of amino acid-producing Escherichia coli using a stoichiometric model and multivariate linear regression.

This work demonstrates a novel computational approach combining flux balance modeling with statistical methods to identify correlations among fluxes in a metabolic network, providing insight as to how the fluxes should be redirected to achieve maximum product yield. The procedure is demonstrated using the example of amino acid production from an industrial Escherichia coli production strain and a hypothetical engineered strain overexpressing two heterologous genes. Regression analysis based on a random sampling of 5,000 points within the feasible solution space of the E. coli stoichiometric network suggested that increased activity of the glyoxylate cycle or PEP carboxylase and elimination of malic enzyme will improve lysine and arginine synthesis.

Amino Acids↗
PubMed1998

Optimization of polyphosphate degradation and phosphate secretion using hybrid metabolic pathways and engineered host strains

Polyphosphate degradation and phosphate secretion were optimized in Escherichia coli strains overexpressing the E. coli polyphosphate kinase gene (ppk) and either the E. coli polyphosphatase gene (ppx) or the Saccharomyces cerevisiae polyphosphatase gene (scPPX1) from different inducible promoters on medium- and high-copy plasmids. The use of a host strain without functional ppk or ppx genes on the chromosome yielded the highest levels of polyphosphate, as well as the fastest degradation of polyphosphate when the gene for polyphosphatase was induced. The introduction of a hybrid metabolic pathway consisting of the E. coli ppk gene and the S. cerevisiae polyphosphatase gene resulted in lower polyphosphate concentrations than when using both the ppk and ppx genes from E. coli, and did not significantly improve the degradation rate. It was also found that the rate of polyphosphate degradation was highest when ppx was induced late in growth, most likely due to the high intracellular polyphosphate concentration. The phosphate released from polyphosphate allowed the growth of phosphate-starved cells; excess phosphate was secreted into the medium, leading to a down-regulation of the phosphate-starvation (Pho) response. The production of alkaline phosphatase, an indicator of the Pho response, can be precisely controlled by manipulating the degree of ppx induction. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗
PubMed2001

Can a 'flawless' live vector vaccine strain be engineered?

The efficiency of any live bacterial vector vaccine hinges on its ability to present sufficient foreign antigen to the human immune system to initiate the desired protective immune response(s). However, synthesis of sufficient levels of heterologous antigen can result in an increase in metabolic burden with an accompanying decrease in the fitness of the live vector, which can ultimately lower desired immune responses to both live vector and heterologous antigen. Here, we explore the underlying mechanisms of metabolic load and propose ways of minimizing such burdens to enhance the fitness and immunogenicity of Salmonella-based live vector vaccines.

Antigens, Bacterial↗
PubMed2006

Genomic inferences of the cis-regulatory nucleotide polymorphisms underlying gene expression differences between Drosophila melanogaster mating races.

Nucleotide sequence polymorphisms affecting gene expression occur in the regulatory region of genes (in cis) and elsewhere in the genome (in trans). Further study is required to weigh the relative importance of cis- and trans-acting mutations in mediating gene expression differences within and between species. Here, microarray hybridization experiments were used to isolate 363 gene expression differences between the female fly head transcriptomes of 2 Drosophila melanogaster strains. One strain (French) represented the cosmopolitan M mating race and the other strain (ZS30) represented the Z mating race derived from Zimbabwe, Africa. From chromosomal substitution strains engineered from the 2 strains, we inferred that the expression differences between M and Z alleles largely could be attributed to the genotype of the chromosomes where the differentially expressed genes were located, that is, cis-regulatory polymorphisms prominently influence gene expression differences between M and Z. The effects of trans-regulatory polymorphisms were apparent yet difficult to quantify. Results have implications for models of gene regulatory evolution as well as experimental studies trying to identify the nucleotide sequence polymorphisms underlying gene expression differences between Drosophila strains.

Animals↗
PubMed2004

Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme.

A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on alpha-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus beta-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of alpha-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of beta-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying beta-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.

Cellulase↗
PubMed1986

The crystallographically determined structures of atypical strained disulfides engineered into subtilisin.

The geometries of two disulfide bridges genetically engineered into subtilisin have been characterized by x-ray crystallography to determine the structural and energetic constraints involved in introducing disulfide bonds into proteins. Both disulfide bridges (Cys-24-Cys-87 and Cys-22-Cys-87) exhibit atypical sets of dihedral angles compared to those for other reported disulfide structures in proteins. The geometric trends for naturally occurring disulfides in protein crystal structures are examined. Comparison of the disulfide-containing mutant protein structures with the wild-type structure shows that, in both cases, disulfide incorporation is accommodated by relatively minor changes in local main-chain conformation. The Cys-22-Cys-87 disulfide has two high energy dihedral angles (X2 = 121 degrees, X2' = 143 degrees). Both disulfides produce short non-bonded contacts with the main-chain.

Bacillus subtilis↗
PubMed1994

Human alpha atrial natriuretic peptide fermentation by using a genetically engineered yeast strain.

The genotype of the recombinant yeast strain Y33::YFD71-3 used in this study is alpha, his, leu, ade and suc. Preliminary batch cultures in shaking flasks showed the expression level of atrial natriuretic peptide (ANP) was 1-2 mg/L. Shaking flask cultures were carried out in YG medium which contained glucose, yeast nitrogen base (YNB), and different amounts of adenine, histidine and leucine. When the growth was limited by adenine, protein secreted from Y33::YFD71-3 cells was increased obviously. Adenine became the limiting substrate when the YG medium was supplemented with 5 g/L of casamino acid (CAA) and the level of ANP expression was influenced by the concentrations of adenine, YNB and leucine in the medium. In fed batch cultures carried out in a RIBE-5 fermenter, the cultures were fed with glucose and a mixture of YNB, CAA, adenine, histidine and leucine to improve cell growth and ANP expression, and the maximum ANP concentration in the culture reached 24.8 mg/L.

Adenine↗
PubMedDate not supplied

Secret signatures inside genomic DNA.

A simple, practical method to watermark short trademarks or signatures into genomic DNA is introduced. Since the marking method is biologically innocuous, it can be applied to all commercialized bacteria to help establish brand names for the engineered strains and to resolve legal disputes regarding gene-related patents. The first such strain of Bacillus subtilis is engineered and is ready to be distributed.

Bacillus subtilis↗
PubMed1999

Cocolonization of the rhizosphere by pathogenic agrobacterium strains and nonpathogenic strains K84 and K1026, used for crown gall biocontrol

The crown gall biocontrol agent strain K84 and three mutants derived from it, K1026 (Tra- deletion mutant of pAgK84), K84 Agr- (lacking pAgK84), and K1143 (lacking pAgK84 and pNoc), significantly reduced gall formation caused by two pathogenic strains resistant to agrocin 84 in peach x almond seedlings planted in infested soil. Cocolonization of roots by pathogenic and nonpathogenic strains was observed in these biocontrol experiments under field conditions. In spite of the efficient biocontrol observed, average populations consisting of 10(2) and 10(6) pathogenic agrobacteria per g of root were found 8 months after planting. The total numbers of pathogenic bacteria on roots were similar for plants treated with the biocontrol strains and for the untreated plants. Strain K84 and the genetically engineered organism K1026 survived at a level of 10(6) agrocin 84-producing bacteria per g of root. The population size of genetically engineered strain K1026 was not significantly different than the population size of wild-type strain K84 8 months after root inoculation. Strains K84 and K1026 controlled two pathogens resistant to agrocin 84 without reducing the total number of pathogenic bacteria in the root system. In addition, this study shows that some biological control activity of strain K84 against agrocin 84-resistant pathogens is independent of plasmids pAgK84 and pNoc.

Journal Article↗
PubMed2006

[Construction of double-labelled carbofuran-degrading bacterium Sphingomonas sp. CDS-1].

The genomic DNA of a carbofuran-degrading bacterium Sphingomonas sp. CDS-1 was digested by Sau3Al and ligated to pRobe-GFP digested by BamHI, and the product was transformed to the E. coli DH5alpha competent cells. Fifty positive clones that could emit green fluorescence under UV were selected from about 1 x 10(4) clones grown on selective plates AmpLB. One clone F7 with the strongest fluorescence was selected, the recombinant plasmid pF7 from this clone was digested with EcoR I & Hind III and the DNA fragment comprising gfp and promoter of Sphingomonas sp. CDS-1 was recovered, which was subsequently cloned into the broad host vector pPZP201 to construct a new plasmid pPZP201-gfp. pPZP201-gfp was introduced into Sphingomonas sp. strain CDS-1 by triparental conjugation to make strain CDS-gfp. gfp was expressed strongly and stably in strain CDS-gfp after 10 times successive re-culturing (48 h/time). The linA gene was inserted into Not I -cut transposon vector pUT/mini-Tn5 to construct a new transposon vector pUT/mini-Tn5-linA. With the aid of helper plasmid pRK600, pUT/mini-Tn5-linA was introduced into CDS-gfp, the dehydrochlorinase gene linA was integrated into the chromosome of CDS-gfp by transposing. The double labelled strain CDS-GFP-LinA was constructed. This strain was also a genetic engineering strain that was able to degrade gamma-hexachlorocyclohexane and carbofuran simultaneously. All of these results laid a foundation for the study of ecological performance of Sphingomonas sp. CDS-1.

Carbofuran↗
PubMed2004

Coherent spin manipulation without magnetic fields in strained semiconductors.

A consequence of relativity is that in the presence of an electric field, the spin and momentum states of an electron can be coupled; this is known as spin-orbit coupling. Such an interaction opens a pathway to the manipulation of electron spins within non-magnetic semiconductors, in the absence of applied magnetic fields. This interaction has implications for spin-based quantum information processing and spintronics, forming the basis of various device proposals. For example, the concept of spin field-effect transistors is based on spin precession due to the spin-orbit coupling. Most studies, however, focus on non-spin-selective electrical measurements in quantum structures. Here we report the direct measurement of coherent electron spin precession in zero magnetic field as the electrons drift in response to an applied electric field. We use ultrafast optical techniques to spatiotemporally resolve spin dynamics in strained gallium arsenide and indium gallium arsenide epitaxial layers. Unexpectedly, we observe spin splitting in these simple structures arising from strain in the semiconductor films. The observed effect provides a flexible approach for enabling electrical control over electron spins using strain engineering. Moreover, we exploit this strain-induced field to electrically drive spin resonance with Rabi frequencies of up to approximately 30 MHz.

Journal Article↗
PubMed2004

Real-time PCR quantification of a green fluorescent protein-labeled, genetically engineered Pseudomonas putida strain during 2-chlorobenzoate degradation in soil.

The potential for real-time PCR (RTm-PCR) detection of the genetically engineered strain Pseudomonas putida GN2 was studied during 2-chlorobenzoate (2-CB) degradation in three different soils. The strain contained the constructed plasmid pGN2 which encoded genes for 2-CB oxidation (cbdA) and the green fluorescent protein (gfp). P. putida GN2 numbers were assessed by plating onto 2-CB minimal media and also by RTm-PCR detection of cbdA and gfp. Addition of P. putida GN2 decreased the time required to degrade 2-CB in all tested soils by more than 7 days. The RTm-PCR estimations of P. putida GN2 numbers strongly correlated with those obtained from plate count methods during active 2-CB degradation. However, after 2-CB degradation in the soils had ceased, RTm-PCR estimations of cbdA and gfp genes were generally one order of magnitude lower than those from plate counts. These results indicate the potential for RTm-PCR to rapidly determine degrader numbers in soil following bioaugmentation but also the need to exercise caution when attempting to determine cell numbers of degraders from the RTm-PCR quantification of plasmid encoded genes after substrate is depleted.

Chlorobenzoates↗
PubMed1999

Glycerol overproduction by engineered saccharomyces cerevisiae wine yeast strains leads to substantial changes in By-product formation and to a stimulation of fermentation rate in stationary phase

Six commercial wine yeast strains and three nonindustrial strains (two laboratory strains and one haploid strain derived from a wine yeast strain) were engineered to produce large amounts of glycerol with a lower ethanol yield. Overexpression of the GPD1 gene, encoding a glycerol-3-phosphate dehydrogenase, resulted in a 1.5- to 2.5-fold increase in glycerol production and a slight decrease in ethanol formation under conditions simulating wine fermentation. All the strains overexpressing GPD1 produced a larger amount of succinate and acetate, with marked differences in the level of these compounds between industrial and nonindustrial engineered strains. Acetoin and 2,3-butanediol formation was enhanced with significant variation between strains and in relation to the level of glycerol produced. Wine strains overproducing glycerol at moderate levels (12 to 18 g/liter) reduced acetoin almost completely to 2,3-butanediol. A lower biomass concentration was attained by GPD1-overexpressing strains, probably due to high acetaldehyde production during the growth phase. Despite the reduction in cell numbers, complete sugar exhaustion was achieved during fermentation in a sugar-rich medium. Surprisingly, the engineered wine yeast strains exhibited a significant increase in the fermentation rate in the stationary phase, which reduced the time of fermentation.

Journal Article↗
PubMed1999

Stable expression of the Lactobacillus casei bacteriophage A2 repressor blocks phage propagation during milk fermentation.

A general strategy was applied to implement resistance against temperate bacteriophages that infect food fermentation starters through cloning and expression of the phage repressor. Lactobacillus casei ATCC 393 and phage A2 were used to demonstrate its feasibility as milk fermentation is drastically inhibited when the strain is infected by this phage. The engineered strain Lact. casei EM40::cI, which has the A2 repressor gene (cI) integrated into the genome, was completely resistant and able to ferment milk whether phage was present or not. In addition, viable phages were eliminated from the milk, probably through adsorption to the cell wall. Finally, the integration of cI in the genome resulted in a stable resistance phenotype, being unnecessary selective pressure during milk fermentation.

Animals↗
PubMed2006

Transcription analysis of recombinant saccharomyces cerevisiae reveals novel responses to xylose.

Lignocellulosic biomass, rich in hexose and pentose sugars, is an attractive resource for commercially viable bioethanol production. Saccharomyces cerevisiae efficiently ferments hexoses but is naturally unable to utilize pentoses. Metabolic engineering of this yeast has resulted in strains capable of xylose utilization. However, even the best recombinant S. cerevisiae strains of today metabolize xylose with a low rate compared to glucose. This study compares the transcript profiles of an S. cerevisiae strain engineered to utilize xylose via the xylose reductase-xylitol dehydrogenase pathway in aerobic chemostat cultures with glucose or xylose as the main carbon source. Compared to the glucose culture, 125 genes were upregulated, whereas 100 genes were downregulated in the xylose culture. A number of genes encoding enzymes capable of nicotinamide adenine dinucleotide phosphate regeneration were upregulated in the xylose culture. Furthermore, xylose provoked increased activities of the pathways of acetyl-CoA synthesis and sterol biosynthesis. Notably, our results suggest that cells metabolizing xylose are not in a completely repressed or in a derepressed state either, indicating that xylose was recognized neither as a fermentable nor as a respirative carbon source. In addition, a considerable number of the changes observed in the gene expression between glucose and xylose samples were closely related to the starvation response.

Acetyl-CoA Carboxylase↗
PubMed2005

[Salmonella choleraesuis C500 delivering DNA immunization against classical swine fever virus].

Classical Swine Fever Virus (CSFV) E2 protein eukaryotic expression plasmid pVAXE2 was constructed. The plasmid pVAXE2 was transformed into Salmonella choleraesuis C500 (S. C500) attenuated vaccine strain by electroporation to generate Salmonella choleraesuis engineering strain S. C500/pVAXE2. The characterization of S. C500/pVAXE2 in morphology, growth, biochemistry and serology indicated that it retained the same properties as its original strain S. C500 with exception of kanamycin resistance originated from the plasmid pVAXE2. The plasmid stable in the bacteria after 15 passages. Kunming mice and rabbits were vaccinated three times at two weeks interval with S. C500/pVAXE2 in oral and intramuscular routes at the dosage of 1 x 10(8) CFU for mice and 2 x 10(9) CFU for rabbits each time. The specific antibody response against CSFV and Salmonella choleraesuis was detected by ELISA. Two weeks after the third boost the immunized rabbits were challenged with 20 ID50 of hog cholera lapinized virus (HCLV), followed by a virulent strain of Salmonella choleraesuis two week later than HCLV challenge. The results showed that all immunized mice and rabbits produced significant antibodies against CSFV and Salmonella choleraesuis, and the immunized rabbits demonstrated the effective protection against the challenge of HCLV and virulent Salmonella choleraesuis. These results indicated the potential of developing multiplex swine DNA vaccine by using this bacteria as the vector.

Animals↗