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PubMed2003

Optimization of biochemical systems by linear programming and general mass action model representations.

A new method is proposed for the optimization of biochemical systems. The method, based on the separation of the stoichiometric and kinetic aspects of the system, follows the general approach used in the previously presented indirect optimization method (IOM) developed within biochemical systems theory. It is called GMA-IOM because it makes use of the generalized mass action (GMA) as the model system representation form. The GMA representation avoids flux aggregation and thus prevents possible stoichiometric errors. The optimization of a system is used to illustrate and compare the features, advantages and shortcomings of both versions of the IOM method as a general strategy for designing improved microbial strains of biotechnological interest. Special attention has been paid to practical problems for the actual implementation of the new proposed strategy, such as the total protein content of the engineered strain or the deviation from the original steady state and its influence on cell viability.

Biochemical Phenomena↗

PubMed2004

[Cloning of a laccase gene from Flammulina velutipes and study on its expression in Pichia pastoris].

Laccase(EC1.10.3.2) can be used for enzymatic detoxification of lignocellulosic hydrolysates. By using molecular techniques such as RACE (rapid amplification of cDNA ends) and Genome-Walking, a laccase gene and its corresponding full-length cDNA were cloned from Flammulina velutipes and designated as glccFv and IccFv. The sequences were submitted to GenBank, and the accession numbers obtained were AY485826 and AY450406, respectively. Analysis of amino acids sequence suggested that one laccase from Polyporus ciliatus possessed the highest homology with the protein encoded by lccFv showing for 72%. The ORF (open reading frame) of lccFv was transformed into Pichia pastoris strain GS115 through the P. pastoris expression vector pHBM906, which contains both the promoter and transcription terminator of the AOX1 gene. The recombinant laccase LCCFv was detected from the engineering strain GS115 (pHBM557) which was fermented with BMMY liquid medium and induced by 1.0% (V/V) methanol at 20 degrees C with the highest expression level (0.1070 U/mL). The optimal reaction temperature of LCCFv that secreted from P. pastoris GS115(pHBM557) was 45 degrees C, the optimal reaction pH value was pH3.9 and the thermostability and pH stability were very well under the optimal conditions.

Base Sequence↗

PubMed2005

Bisphosphonate inhibitors of Toxoplasma gondi growth: in vitro, QSAR, and in vivo investigations.

We have investigated the activity of 60 bisphosphonates against the replication of Toxoplasma gondii in vitro and of three of the most active compounds, in vivo. The two most active compounds found were n-alkyl bisphosphonates containing long (n = 9 or 10) hydrocarbon chains, not the nitrogen-containing species used in bone resorption therapy. The target of all of the most active bisphosphonates appears to be the isoprene biosynthesis pathway enzyme farnesyl pyrophosphate synthase (FPPS), as indicated by the correlations between T. gondii growth inhibition and FPPS (human and Leishmania major) enzyme inhibition and by the fact that a T. gondii strain engineered to overexpress FPPS required considerably higher levels of bisphosphonates to achieve 50% growth inhibition, while the IC(50) for atovaquone (which does not inhibit FPPS) remained the same in the overexpressing strain. The phosphonate inhibitor of the non-mevalonate pathway, fosmidomycin, which inhibits the enzyme 1-deoxyxylulose-5-phosphate reductoisomerase, had no effect on T. gondii growth. To investigate structure-activity relationships (SARs) in more detail, we used two three-dimensional quantitative SAR methods: comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), to investigate all 60 bisphosphonates. Both the CoMFA and CoMSIA models indicated a 60-70% contribution from steric interactions and a 30-40% contribution from electrostatic interactions and using four N = 55 training sets for each method, we found on average between a factor of 2 and 3 error in IC(50) prediction. The three most active compounds found in vitro were tested in vivo in a Smith-Webster mouse model and the two most active bisphosphonates were found to provide up to an 80% protection from death, a considerable improvement over that found previously with nitrogen-containing bisphosphonates. This effect may originate in the much higher therapeutic indices of these alkyl bisphosphonates, as deduced from in vitro assays using LD(50) values for growth inhibition of a human cell line. Overall, these results indicate that alkyl bisphosphonates are promising compounds for further development as agents against Toxoplasma gondii growth, in vivo.

Aldose-Ketose Isomerases↗

PubMed2006

Exploitation of a beta-lactamase reporter gene fusion in the carbapenem antibiotic production operon to study adaptive evolution in Erwinia carotovora.

Erwinia carotovora subsp. carotovora strain ATTn10 produces the beta-lactam antibiotic 1-carbapen-2-em-3-carboxylic acid (carbapenem) by expressing the carABCDEFGH operon. Mutants exhibiting increased carbapenem gene transcription were positively selected using an engineered strain with a functional beta-lactamase translational fusion in carH, the last gene of the operon. However, spontaneous ampicillin-resistant mutants were isolated even when transcription of carH : : blaM was blocked by a strongly polar mutation in carE. The mechanism of resistance was shown to be due to cryptic IS10 elements transposing upstream of carH : : blaM, thereby providing new promoters enabling carH : : blaM transcription. Southern blots showed that IS10 was present in multicopy in ATTn10. In addition, a Tn10 genetic remnant was discovered. The results offer insights into the genetic archaeology of strain ATTn10 and highlight the powerful impacts of cryptic IS elements in bacterial adaptive evolution.

Adaptation, Biological↗

PubMed2004

Rapid preparation of isotopolog libraries by in vivo transformation of 1)C-glucose. Studies on 6,7-dimethyl-8-ribityllumazine, a biosynthetic precursor of vitamin B2.

An Escherichia coli strain engineered for expression of the ribABGH genes of Bacillus subtilis was shown to produce 100 mg of the riboflavin precursor 6,7-dimethyl-8-ribityllumazine per liter of minimal medium. Growth of the recombinant strain in medium supplemented with [U-13C6]glucose and/or 15NH4Cl as single sources of carbon and/or nitrogen afforded 6,7-dimethyl-8-ribityllumazine universally labeled with 13C and/or 15N. The yield of [U-13C13]-6,7-dimethyl-8-ribityllumazine based on [U-13C6]glucose was 25 mg/g. Fermentation with [1-13C1]-, [2-13C1]-, or [3-13C1]glucose afforded mixtures of 6,7-dimethyl-8-ribityllumazine isotopologs, predominantly with 13C enrichment of single carbon atoms. The isotope-labeled samples enabled a comprehensive NMR analysis of 6,7-dimethyl-8-ribityllumazine. Isotopolog libraries of a wide variety of microbial metabolites can be produced by the same experimental approach.

Bacillus subtilis↗

PubMed1995

Heterozygosity at the b mating-type locus attenuates fusion in Ustilago maydis.

Mating and pathogenesis of the corn smut fungus, Ustilago maydis, are controlled by two unlinked mating-type loci, a and b. Yeast-like haploids that differ at both loci are compatible and fuse to establish a pathogenic dikaryon. Mating is assayed in vitro by co-inoculation on culture medium containing activated charcoal; compatible combinations have a characteristic "fuzzy" appearance caused by the growth of aerial hyphae. In general, this test has not been useful for assaying the mating ability of strains that are already mycelial (e.g., those heterozygous at b or at both mating-type loci). Using an assay for cytoduction involving transfer of a mitochondrial marker during transient cell fusion, and engineered strains with defined genotypes, we examined the mating abilities of strains heterozygous or hemizygous at the mating-type loci. The data (which have not been available from conventional pathogenicity or plate mating tests) show that heterozygosity at b attenuates fusion in haploid and diploid strains, whereas strains heterozygous at a retain the ability to fuse with a compatible haploid partner. It appears, therefore, that subsequent fusion events are attenuated once fusion has occurred to establish the U. maydis dikaryon.

Cell Fusion↗

PubMed2003

[Genetically marking of natural biocontrol bacterium Bacillus subtilis strains with green fluorescent protein gene].

The full length sequence of the promoter and gfp gene were obtained respectively by PCR with two pairs unique primers PxyF/R and primers gfpF/R, which were designed according to the gfp gene and promoter sequence of xylase operon from Bacillus subtilis 168, and the DNA template plasmids pHT315-xyIR and pGFPuv. Furthermore, the fused translational expression cassette PxylR-gfp was constructed using overlapping PCR technique with the primers pair PxyF/gfpR and the mixture of above PCR production. After being digested by Kpn I and Sph I , PxylR-gfp expression cassette was inserted into E. coli-B. thuringiensis shuttle vecter pHT315 and E. coli-B. subtilis shuttle vecter pRP22, and the resulted recombinant plasmids were named as pGFP315 and pGFP22 respectively. Both recombinant plasmids were transferred into B. subtilis lab strain 168 and the resulted transformants are bright green performance under 365 nm UV light. However, only pGFP22 can be introduced into the natural strain B916. The transformants containing pGFP22 have bright green performance under 365 nm UV light and was named B916-gfp. Antifungal activities testing results proved that there is no obvious difference between B916 and the engineered strains B916-gfp. Research results also showed that the stability of B916-gfp was 94% after growth about 175 generations at 37 degrees C, and the losing rate of plasmid was less than 3.5 x 10(-4) per generation.

Bacillus subtilis↗

PubMed1993

Cloning, sequencing and enhanced expression of the Trichoderma reesei endoxylanase II (pI 9) gene xln2.

The Trichoderma reesei xln2 gene coding for the pI9.0 endoxylanase was isolated from the wild-type strain QM6a. The gene contains one intron of 108 nucleotides and codes for a protein of 223 amino acids in which two putative N-glycosylation target sites were found. Three different T. reesei strains were transformed by targeting a construct composed of the xln2 gene, including its promoter, to the endogenous cbh1 locus. Highest overall production levels of xylanase were obtained using T. reesei ALKO2721, a genetically engineered strain, as a host. Integration into the cbh1 locus was not required for enhanced expression under control of the xln2 promoter.

Amino Acid Sequence↗

PubMed2001

Coexpression of chitinase and the cry11Aa1 toxin genes in Bacillus thuringiensis serovar israelensis.

At the spore stage, a cloned chitinase gene was coexpressed with the regulatory gene p19 and the toxin gene cry11Aa1 in the hosts Bacillus thuringiensis serovar israelensis strains 4Q2-72 and c4Q2-72. The chitinase gene was derived from a high-chitinase producer, Bacillus licheniformis TP-1. Two transcriptional fusion plasmids between the p19 or p19-cry11Aa1 genes and the promoterless chitinase gene were constructed. In transcription order, the p16-19CHI construct contained the p19 gene together with the chitinase gene only while the p16-1968CHI construct contained p19 together with the toxin gene cry11Aa1 and the chitinase gene. The inserted sequences were regulated by a spore-specific promoter located upstream of p19. The recombinant chitinase of all transformed B. thuringiensis serovar israelensis strains was initially synthesized at low level at about 9 h of growth when a portion of the cells started to sporulate. It increased thereafter and reached maximum levels of 5.5, 4.9, and 4.7 mU/ml at 48 h, for strain 4Q2-72 transformed with p16-19CHI and p16-1968CHI and strain c4Q2-72 transformed with p16-19CHI, respectively. This activity was approximately 2 times higher than the maximum activity (2.7 mU/ml) of the parental strain, B. licheniformis TP-1. Although crude chitinase alone from B. thuringiensis serovar israelensis c4Q2-72 (p16-19CHI) at 4.5 mU/ml caused 40% mortality in second instar Aedes aegypti larvae, transformants containing the chitinase alone or in combination with cry11Aa1 resulted in lower toxicity to A. aegypti larvae than the untransformed 4Q2-72 host. For example the LC(50) for the transformed 4Q2-72 harboring the chitinase gene only (p16-19CHI) was 5.6 x 10(4) +/- 0.7 x 10(4) cells, 40 times higher than that of the untransformed host at 1.4 x 10(3) +/- 0.19 x 10(3). The lower toxicity correlated with poor sporulation in the transformants (i.e., 35 times lower than that in the untransformed host). However, the transformed 4Q2-72 strain expressing both the chitinase and the cry11Aa1 toxin genes (p16-1968CHI) were only 4-fold less toxic (LC(50) = 5.6 x 10(3) +/- 1.99 x 10(3)) than the untransformed 4Q2-72 hosts even though their spore count was 300 times lower. Since coapplication of crude chitinase from the cloned gene in recombinant strain (c4Q2-72 harboring p16-19CHI) with cell suspensions of B. thuringiensis serovar israelensis 4Q2-72 and its transformants could enhance 3- to 50-fold larvicidal activity, improvement in sporulation ability of these genetically engineered strains and cocrystallization of chitinase with crystal toxins may increase their potential for future insect control.

Aedes↗

PubMedDate not supplied

Development of vaccines for bacterial diseases using recombinant DNA technology.

A vaccine was prepared using recombinant DNA techniques to prevent fatal enterotoxigenic Escherichia coli diarrhea in swine. The product, which is a subunit vaccine, was prepared by mechanical and chemical removal of pilus adhesins from the surface of genetically engineered strains of E. coli. The vaccine contains the pilus adhesins K88, K99, and 987P plus an adjuvant. The genes responsible for production of K88 and K99 were separately cloned into the multicopy vector pBR322. K88 was found to be encoded on a 7.6-kilobase HindIII-EcoRI fragment, and K99 was found to be encoded on a 7.15-kilobase BamHI fragment. Strains containing the recombinant plasmid for K99 produced up to ten times more K99 than strains containing the wild-type plasmid. Vaccination of pregnant pigs with the vaccine led to production of pilus-adhesin-specific antibodies that were transferred to the piglets in colostrum and milk. Pilus-adhesin-specific antibodies neutralized the adhesiveness of the pili on enterotoxigenic E. coli, thus preventing attachment, colonization, and disease. Mortality of pigs in litters from vaccinated pigs due to experimentally induced enterotoxigenic E. coli diarrhea was reduced 10-to-20-fold (depending upon the challenge strain), and the incidence, severity, and duration of diarrhea were also reduced.

Animals↗

PubMed2004

Analysis of relative levels of production of pertussis toxin subunits and Ptl proteins in Bordetella pertussis.

Pertussis toxin is transported across the outer membrane of Bordetella pertussis by the type IV secretion system known as the Ptl transporter, which is composed of nine different proteins. In order to determine the relative levels of production of pertussis toxin subunits and Ptl proteins in B. pertussis, we constructed translational fusions of the gene for alkaline phosphatase, phoA, with various ptx and ptl genes. Comparison of the alkaline phosphatase activity of strains containing ptx'- or ptl'-phoA fusions indicated that pertussis toxin subunits are produced at higher levels than Ptl proteins, which are encoded by genes located toward the 3' end of the ptx-ptl operon. We also engineered strains of B. pertussis by introducing multiple copies of the ptl genes or subsets of these genes and then examined the ability of each of these strains to secrete pertussis toxin. From these studies, we determined that certain Ptl proteins appear to be limiting in the secretion of pertussis toxin from the bacteria. These results represent an important first step in assessing the stoichiometric relationship of pertussis toxin and its transporter within the bacterial cell.

Alkaline Phosphatase↗

PubMed2006

Quantitative in situ assay of salicylic acid in tobacco leaves using a genetically modified biosensor strain of Acinetobacter sp. ADP1.

Salicylic acid (SA) plays important roles in plants, most notably in the induction of systemic acquired resistance (SAR) against pathogens. A non-destructive in situ assay for SA would provide new insights into the functions of SA in SAR and other SA-regulated phenomena. We assessed a genetically engineered strain of Acinetobacter sp. ADP1, which proportionally produces bioluminescence in response to salicylates including SA and methylsalicylate, as a reporter for salicylate accumulation in the apoplast of plant leaves. SA was measured quantitatively in situ in NN genotype tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves inoculated with tobacco mosaic virus (TMV). The biosensor revealed accumulation of apoplastic SA before the visible appearance of hypersensitive response (HR) lesions. When the biosensor was infiltrated into TMV-inoculated leaves displaying HR lesions at 90 and 168 h post-inoculation, salicylate accumulation was detected predominantly in tissues surrounding the lesions and in veins adjacent to HR lesions. These images are consistent with previous data demonstrating that SA accumulation occurs prior to and following the onset of visible HR lesions. We also used the biosensor to observe apoplastic SA accumulation in tobacco leaves inoculated with virulent and HR-eliciting strains of the bacterial plant pathogen Pseudomonas syringae. The work demonstrates that the Acinetobacter sp. ADP1 biosensor is a useful new tool to non-destructively assay salicylates in situ and to map their spatial distribution in plant tissues.

Acinetobacter↗

PubMed1996

Differential regulation of Ia expression and antigen presentation by listeriolysin-producing versus non-producing strains of Listeria monocytogenes.

Listeria monocytogenes is an intracellular bacterial pathogen. A single gene product, listeriolysin (LLO), is critical for the induction of protective immunity. We now show that listeria that produce functional LLO augment Ia expression by macrophages and are better presented to a Th1, CD4+ anti-listeria T cell line. We used two genetically engineered strains of listeria which differed only in their ability (Ly+) or inability (Ly-) to produce functional LLO. Ia-negative murine macrophages ingested either Ly+ or Ly-, and then were stimulated by interferon-gamma (IFN-gamma). Increasing numbers of live Ly+, but not Ly-, augmented IFN-gamma-induced Ia expression. Ly+ by itself did not induce Ia expression. Heat-killed Ly+ and Ly- did not augment IFN-gamma-induced Ia expression. The abundance of Ia on the macrophage cell surface is one major determinant of antigen presentation to CD4+ T cells. Consistent with their ability to augment la expression, Ly+ were better presented than Ly- to a CD4+, Th1, anti-listeria T cell line. When macrophages and T cells were from different inbred mouse strains, antigen presentation required identity at the class II region of the MHC gene complex. This indicated that antigen presentation occurred via Ia molecules. The increased ability of macrophages to present Ly+ is a product of the macrophage-listeria interaction, not a property of the T cell tine 86. If Ia-negative macrophages ingested Listeria and were then stimulated by IFN-gamma, Ly+ was presented more efficiently than Ly-. On the other hand, if Ia-positive macrophages ingested Listeria, then Ly+ and Ly- were presented equally well to T cells. Altogether our data is consistent with the hypothesis that macrophages interact differently with Ly+, and that this contributes to the ability of only live Ly+ to induce protective immunity.

Animals↗

PubMed1983

Anaerobic fermentations--some new possibilities.

Anaerobic fermentations start with the major advantage that, unlike aerobic processes, they do not incur the cost penalties associated with the need to sustain high rates of culture aeration, agitation and cooling. Even so, many of the more traditional fermentations that yield high volume, low value products (such as alcohols or fatty acids) are currently only economically viable if they utilize biological wastes and agricultural surpluses as substrates. To achieve maximum conversion efficiency it is necessary to select the appropriate species/strain of micro-organism which will perform best under the intended conditions of operation (which could include elevated temperatures and extremes of pH). Knowledge of the mechanisms whereby the fermentation is physiologically controlled can then suggest means, including genetically engineered strain improvements and/or the establishment of stable co-cultures with other microbes, whereby the yield of a desired product can be optimized. The biology of a fermentation process can thus be rendered sufficiently consistent and predictable for the biochemical engineer to be able to achieve optimal fermenter productivity. Anaerobic processes are particularly well suited to a continuous flow mode of operation (with cell retention) and new techniques for selective product removal and concentration can reduce the energy costs associated with 'downstream processing'. Hitherto, most interest has been centred on those anaerobic fermentations of renewable biomass which yield immediately useful products (fuel alcohols, methane). However, by combining biological and chemical procedures the profitability of even some superficially unpromising fermentations could be substantially enhanced. Collaborative research by microbial physiologists, geneticists and biochemical engineers is a prerequisite for marketable success in new applications of fermentative anaerobes and their enzymes.

Anaerobiosis↗

PubMed1995

Differential regulation of TNF-alpha production by listeriolysin-producing versus nonproducing strains of Listeria monocytogenes.

Only Listeria monocytogenes that produce listeriolysin O (LLO) elicit protective immunity. Given the importance of tumor necrosis factor alpha (TNF-alpha) in anti-Listeria immunity, we have investigated TNF-alpha production by macrophages after they ingested live LLO-producing compared to LLO-non-producing bacteria. We used two genetically engineered strains of Listeria that differed only in their ability (Ly+) or inability (Ly-) to produce LLO. Ly+ and Ly- caused the same kinetics of increased mRNA abundance for TNF-alpha during the first 90 min after phagocytosis. However, only Ly+ caused sustained transcription of TNF-alpha mRNA, and this may account for the increased release of TNF-alpha. The transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) prevented the sustained abundance of cytokine mRNA 20 h after ingestion of Ly+. In addition, nuclear run-on assays indicated sustained transcription of TNF-alpha genes only after ingestion of Ly+. LLO itself was not responsible for the ability of Ly+ to stimulate the sustained transcription of the TNF-alpha genes. Instead, LLO may allow Listeria to survive within macrophages so that other bacterial products cause sustained TNF-alpha gene transcription. Both Ly+ and Ly- produced molecules, isolated by 50% ammonium sulfate, that induced cytokine production. In conclusion, we now report that Ly+ causes sustained transcription of the TNF-alpha gene and production of TNF-alpha by macrophages in vitro. We speculate that the TNF-alpha may activate endothelium and thus allow the recruitment of T cells to sites of infection. This may contribute to the ability of only LLO-producing Listeria to induce protective immunity.

Animals↗

PubMed2006

Visualizing fungal infections in living mice using bioluminescent pathogenic Candida albicans strains transformed with the firefly luciferase gene.

Animal studies with Candida albicans have provided models for understanding fungal virulence and antifungal drug development. To non-invasively monitor long-term Candida murine infections, clinical isolates were stably transformed with a codon-optimized luciferase gene to constitutively express luciferase. Chronic systemic infections were established in mice with engineered strains, and bioluminescent signals were apparent from kidneys by non-invasive imaging using charged-coupled device cameras. These infections were established in immune-competent mice, and bioluminescence was detectable in animals that showed no physiological consequence of infection, as well as those visually succumbing to the disease. Similarly, bioluminescence was measured from the vaginal tissue of mice infected vaginally. Fungal loads determined by plating vaginal lavages showed a similar pattern to the bioluminescent signals measured, and fungal infection could be detected in animals for over 30 days post infection by both modalities. The effect of the antifungal drug miconazole was tested in this model, and clearance in animals was apparent by both direct imaging and fungal load determination. The use of bioluminescence to monitor these and other models of Candida infections will greatly speed up the analysis of drug development studies, both in ease of visualizing infections and decreasing numbers of animals required to run such studies.

Animals↗

PubMedDate not supplied

Control of secondary metabolite congener distributions via modulation of the dissolved oxygen tension.

Many secondary metabolites, including various polyketides, require complex enzymatic pathways for modification into their final biologically active forms. Limitation of the dissolved oxygen supplied during cultivation of various microbial strains can decrease the activity of cytochrome P-450 monooxygenases required for the processing of pathway intermediates into their final forms, resulting in the accumulation of these intermediates as the primary products. Here, a generalized oxygen-limited cultivation strategy is specifically demonstrated with a myxobacterial strain engineered to heterologously express the epothilone polyketide synthase (PKS) gene cluster under either an excess (the dissolved oxygen tension is maintained at 50% of saturation) or a depleted (no residual dissolved oxygen detected) level of oxygenation during cultivation. Cultivation of this myxobacterial strain with excess oxygenation resulted in the production of epothilones A and B as the primary products, while cultivation of this same strain under depleted oxygenation resulted in the production of epothilones C and D as the primary products. Additionally, the peak cell density in the oxygen-depleted cultivations was 60% higher than that observed in oxygen-excess cultivations. Finally, an active EpoK epoxidase was found to catalyze the production of a novel epothilone (Epo506) with an unexpected structure during the cultivation of another myxobacterial strain expressing a genetically modified epothilone PKS under excess oxygenation. The structure of Epo506 was determined by high-resolution mass spectrometry and one- and two-dimensional NMR.

Bioreactors↗

PubMed1995

Effects of Bacillus subtilis sporulation regulatory protein SpoIIID on transcription by sigma K RNA polymerase in vivo and in vitro.

SpoIIID is a sequence-specific, DNA-binding protein that activates or represses transcription of different genes by sigma K RNA polymerase in vitro. A Bacillus subtilis strain engineered to produce both sigma K and SpoIIID during growth showed effects of SpoIIID on expression of sigma K-dependent genes that were consistent with the effects of a small amount of SpoIIID on transcription of these genes in vitro, indicating that the strain provides a simple, in vivo method to screen for effects of SpoIIID on transcription of sigma K-dependent genes.

Bacillus subtilis↗