SEARCH · PubMed Health

Results for “strain engineering”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 235 records · Page 13Linked to original sources

PubMed2006

Heterologous expression of tylosin polyketide synthase and production of a hybrid bioactive macrolide in Streptomyces venezuelae.

Tylosin polyketide synthase (Tyl PKS) was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin PKS gene cluster using two compatible low-copy plasmids, each under the control of a pikAI promoter. The mutant strain produced 0.5 mg/l of the 16-membered ring macrolactone, tylactone, after a 4-day culture, which is a considerably reduced culture period to reach the maximum production level compared to other Streptomyces hosts. To improve the production level of tylactone, several precursors for ethylmalonyl-CoA were fed to the growing medium, leading to a 2.8-fold improvement (1.4 mg/ml); however, switching the pikAI promoter to an actI promoter had no observable effect. In addition, a small amount of desosamine-glycosylated tylactone was detected from the extract of the mutant strain, revealing that the native glycosyltransferase DesVII displayed relaxed substrate specificity in accepting the 16-membered ring macrolactone to produce the glycosylated tylactone. These results demonstrate a successful attempt for a heterologous expression of Tyl PKS in S. venezuelae and introduce S. venezuelae as a rapid heterologous expression system for the production of secondary metabolites.

Anti-Bacterial Agents↗

PubMedDate not supplied

Photosynthetic accumulation of poly-(hydroxybutyrate) by cyanobacteria--the metabolism and potential for CO2 recycling.

Regulatory mechanism in PHB [poly-(hydroxybutyrate)] accumulation by cyanobacteria, especially by a thermophilic isolate, Synechococcus MA19 was reviewed in comparison with a genetically engineered strain. The strain, MA19 accumulates PHB under nitrogen starved and photoautotrophic conditions (MA19-N). Little PHB synthase activity was detected in crude extracts from the cells grown in nitrogen sufficient conditions (MA19 + N). The activity was detected exclusively in membrane fractions from MA19 + N. The change of the enzyme activity was insensitive to chloramphenicol, which suggests post-translational activation. In vitro, acetyl phosphate activated PHB synthase in membrane fractions from MA19 + N, and the extent of activation depended on the concentration of acetyl phosphate. Phosphotransacetylase which catalyzes the conversion of acetyl-CoA to acetyl phosphate was detected in crude extracts from MA19-N but not in those from MA19 + N. These results suggested that intracellular acetyl phosphate concentration could be controlled, depending on C-N balance and intracellular acetyl-CoA concentration. On the contrary, in genetically-engineered cyanobacterium (transformant with PHB synthesizing genes from Ralstonia eutropha), it did not seem to be PHB synthase but acetyl-CoA flux that limits PHB synthesis. The closer association of PHB granules with thylakoid membranes in MA19 is suggested than that in the genetically-engineered cyanobacterium, which may reflect the difference of distribution of PHB synthase. Transposon-mutagenesis was used to acquire mutants of its altered PHB regulatory mechanism. PHA production by cyanobacteria was considered from the aspects of photobioreactors.

Acyltransferases↗

PubMed2002

Functional expression of genes involved in the biosynthesis of the novel polyketide chain extension unit, methoxymalonyl-acyl carrier protein, and engineered biosynthesis of 2-desmethyl-2-methoxy-6-deoxyerythronolide B.

A subcluster of five genes, asm13-17, from the ansamitocin biosynthetic gene cluster of Actinosynnema pretiosum was coexpressed in Streptomyces lividans with the genes encoding the 6-deoxyerythronolide B (6-DEB) synthase from Saccharopolyspora erythraea, in which the methylmalonate-specifying AT6 domain had been replaced by the methoxymalonate-specifying AT8 domain from the FK520 cluster of Streptomyces hygroscopicus. The engineered strain produced the predicted product, 2-desmethyl-2-methoxy-DEB, instead of 6-DEB and 2-desmethyl-6-DEB, which were formed in the absence of the asm13-17 cassette, indicating that asm13-17 are sufficient for synthesis of this unusual chain extension unit. Deletion of asm17, encoding a methyltransferase, from the cassette gave 6-DEB instead of its hydroxy analogue, indicating that methylation of the extender unit is required for its incorporation.

Acyl Carrier Protein↗

PubMed1997

Fermentation of biomass-derived glucuronic acid by pet expressing recombinants of E. coli B.

The economics of large-scale production of fuel ethanol from biomass and wastes requires the efficient utilization of all the sugars derived from the hydrolysis of the heteropolymeric hemicellulose component of lignocellulosic feedstocks. Glucuronic and 4-O-methyl-glucuronic acids are major side chains in xylans of the grasses and hardwoods that have been targeted as potential feedstocks for the production of cellulosic ethanol. The amount of these acids is similar to that of arabinose, which is now being viewed as another potential substrate in the production of biomass-derived ethanol. This study compared the end-product distribution associated with the fermentation of D-glucose (Glc) and D-glucuronic acid (GlcUA) (as sole carbon and energy sources) by Escherichia coli B (ATCC 11303) and two different ethanologenic recombinants--a strain in which pet expression was via a multicopy plasmid (pLOI297) and a chromosomally integrated construct, strain KO11. pH-stat batch fermentations were conducted using a modified LB medium with 2% (w/v) Glc or GlcUA with the set-point for pH control at either 6.3 or 7.0. The nontransformed host culture produced only lactic acid from glucose, but fermentation of GlcUA yielded a mixture of ethanol, acetic, and lactic acids, with acetic acid being the predominant end-product. The ethanol yield associated with GlcUA fermentation by both recombinants was similar, but acetic acid was a significant by-product. Increasing the pH from 6.3 to 7.0 increased the rate of glucuronate fermentation, but it also decreased the ethanol mass yield from 0.22 to 0.19 g/g primarily because of an increase in acetic acid production. In all fermentations there was good closure of the carbon mass balance, the exception being the recombinant bearing plasmid pLOI297 that produced an unidentified product from GlcUA. The metabolism of GlcUA by this metabolically engineered construct remains unresolved. The results offered insights into metabolic fluxes and the regulation of pyruvate catabolism in the wild-type and engineered strains. End-product distribution for metabolism of glucuronic acid by the nontransformed, wild-type E. coli B and recombinant strain KO11 suggests that the enzyme pyruvate-formate lyase is not solely responsible for the production of acetylCoA from pyruvate and that derepressed pyruvate dehydrogenase may play a significant role in the metabolism of GlcUA.

Acetyltransferases↗

PubMed2005

A fast and flexible PEG-mediated transient expression system in plants for high level expression of secreted recombinant proteins.

Plant expression systems offer a valuable alternative to traditional systems for the production of recombinant biopharmaceuticals. A highly efficient polyethyleneglycol (PEG)-mediated transient expression system for secreted recombinant proteins in plants has been developed. The human vascular endothelial growth factor 121 (rhVEGF) has been successfully expressed and efficiently secreted into the culture medium by transiently transformed moss protoplasts. In order to obtain secretion efficiency data, different expressed signal peptides were analysed and time course studies were performed with expression constructs containing different promoters. The transformation procedure was optimised for high level expression (up to 10 microg/ml) and successfully performed even with a transgenic glyco-engineered strain lacking plant-specific immunogenic sugar residues in N-glycans. The amount of rhVEGF was produced in such quantity that it allowed for the analysis of biological activity, silver-staining and Western blotting, revealing the correct formation and processing of the homodimer. This fast and flexible transient expression system enables feasibility studies and construct optimisation to be concluded within a few days, thus avoiding the time consuming step of having to generate stably transformed lines.

Bryopsida↗

PubMed2002

Novel Saccharomyces cerevisiae screen identifies WR99210 analogues that inhibit Mycobacterium tuberculosis dihydrofolate reductase.

The ongoing selection of multidrug-resistant strains of Mycobacterium tuberculosis has markedly reduced the effectiveness of the standard treatment regimens. Thus, there is an urgent need for new drugs that are potent inhibitors of M. tuberculosis, that exhibit favorable resistance profiles, and that are well tolerated by patients. One promising drug target for treatment of mycobacterial infections is dihydrofolate reductase (DHFR; EC 1.5.1.3), a key enzyme in folate utilization. DHFR is an important drug target in many pathogens, but it has not been exploited in the search for drugs effective against M. tuberculosis. The triazine DHFR inhibitor WR99210 has been shown to be effective against other mycobacteria. We show here that WR99210 is also a potent inhibitor of M. tuberculosis and Mycobacterium bovis BCG growth in vitro and that resistance to WR99210 occurred less frequently than resistance to either rifampin or isoniazid. Screening of drugs with M. tuberculosis cultures is slow and requires biosafety level 3 facilities and procedures. We have developed an alternative strategy: initial screening in an engineered strain of the budding yeast Saccharomyces cerevisiae that is dependent on the M. tuberculosis DHFR for its growth. Using this system, we have screened 19 compounds related to WR99210 and found that 7 of these related compounds are also potent inhibitors of the M. tuberculosis DHFR. These studies suggest that compounds of this class are excellent potential leads for further development of drugs effective against M. tuberculosis.

Antibiotics, Antitubercular↗

PubMed2001

Nitric oxide signaling and transcriptional control of denitrification genes in Pseudomonas stutzeri.

The expression of denitrification by a facultatively anaerobic bacterium requires as exogenous signals a low oxygen tension concomitant with an N oxide. We have studied the role of nitric oxide (NO), nitrous oxide (N2O), and nitrite as signal molecules for the expression of the denitrification apparatus of Pseudomonas stutzeri. Transcriptional kinetics of structural genes were monitored by Northern blot analysis in a 60-min time frame after cells were exposed to an N oxide signal. To differentiate the inducer role of NO from that of nitrite, mRNA kinetics were monitored under anoxic conditions in a nirF strain, where NO generation from nitrite is prevented because of a defect in heme D(1) biosynthesis. NO-triggered responses were monitored from the nirSTB operon (encoding cytochrome cd(1) nitrite reductase), the norCB operon (encoding NO reductase), nosZ (encoding nitrous oxide reductase), and nosR (encoding a putative regulator). Transcription of nirSTB and norCB was activated by 5 to 50 nM NO, whereas the nosZ promoter required about 250 nM. Nitrite at 5 to 50 nM elicited no response. At a threshold concentration of 650 nM N2O, we observed in the anoxic cell the transient appearance of nosZ and nosR transcripts. Constant levels of transcripts of both genes were observed in an anoxic cell sparged with N2O. NO at 250 nM stimulated in this cell type the expression of nos genes severalfold. The transcription factor DnrD, a member of the FNR-CRP family, was found to be part of the NO-triggered signal transduction pathway. However, overexpression of dnrD in an engineered strain did not result in NirS synthesis, indicating a need for activation of DnrD. NO modified the transcriptional pattern of the dnrD operon by inducing the transcription of dnrN and dnrO, located upstream of dnrD. Insertional mutagenesis of dnrN altered the kinetic response of the nirSTB operon towards nitrite. Our data establish NO and DnrD as key elements in the regulatory network of denitrification in P. stutzeri. The NO response adds to the previously identified nitrate-nitrite response mediated by the NarXL two-component system for the expression of respiratory nitrate reductase encoded by the narGHJI operon.

Gene Expression Regulation, Bacterial↗

PubMed2001

The endosymbionts of tsetse flies: manipulating host-parasite interactions.

Through understanding the mechanisms by which tsetse endosymbionts potentiate trypanosome susceptibility in tsetse, it may be possible to engineer modified endosymbionts which, when introduced into tsetse, render these insects incapable of transmitting parasites. In this study we have assayed the effect of three different antibiotics on the endosymbiotic microflora of tsetse (Glossina morsitans morsitans). We showed that the broad-spectrum antibiotics, ampicillin and tetracycline, have a dramatic impact on tsetse fecundity and pupal emergence, effectively rendering these insects sterile. This results from the loss of the tsetse primary endosymbiont, Wigglesworthia glossinidia, which is eradicated by ampicillin and tetracycline treatment. Using the sugar analogue and antibiotic, streptozotocin, we demonstrated specific elimination of the tsetse secondary endosymbiont, Sodalis glossinidius, with no observed detrimental effect upon W. glossinidia. The specific eradication of S. glossinidius had a negligible effect upon the reproductive capability of tsetse but did effect a significant reduction in fly longevity. Furthermore, elimination of S. glossinidius resulted in increased refractoriness to trypanosome infection in tsetse, providing further evidence that S. glossinidius plays an important role in potentiating trypanosome susceptibility in this important disease vector. In the light of these findings, we highlight progress made towards developing recombinant Sodalis strains engineered to avoid potentiating trypanosome susceptibility in tsetse. In particular, we focus on the chitinase/N-acetyl-D-glucosamine catabolic machinery of Sodalis which has previously been implicated in causing immune inhibition in tsetse.

Acetylglucosamine↗

PubMed1994

Engineered biosynthesis of novel polyketides: evidence for temporal, but not regiospecific, control of cyclization of an aromatic polyketide precursor.

BACKGROUND: Aromatic polyketide synthases (PKSs) catalyze the formation and cyclization of polyketide chains of variable lengths, generating a family of compounds of proven medical significance. Initial control over the regiospecificity of cyclization is believed to be exercised by the minimal PKS, composed of the three essential components for polyketide biosynthesis, which catalyzes an intramolecular aldol condensation towards the middle of the chain. Subsequent cyclization reactions are either catalyzed by additional components of the PKS, or occur in the absence of specific catalysts. RESULTS: Structural and biosynthetic studies on SEK4b, a novel octaketide product of a minimal PKS, revealed an unusual cyclization pattern. The first cyclization (an aldol condensation) occurs at the methyl end of the unreduced polyketide backbone precursor. This is followed by hemiketal formation and lactonization. The overall structure of SEK4b is similar to that of SEK4, a previously-identified product of the same genetically-engineered strain, differing only in the positions of a methyl and a pyrone group around a common fused-ring system. The biosynthetic pathways of the two molecules are quite different, however. The yield of SEK4b relative to SEK4 is much higher in the absence of PKS components (aromatases and cyclases) acting later in the pathway. CONCLUSIONS: In this cyclization pathway, the regiospecificity of cyclization is not directly controlled by the minimal PKS. Instead, we propose that the enzyme influences cyclization by controlling the timing of chain release. Chain release and cyclization may be concurrent with synthesis. Other PKS subunits appear to stabilize the complex of the PKS with the nascent chain, preventing premature release.

Catalysis↗

PubMed2006

Directed evolution of RuBisCO hypermorphs through genetic selection in engineered E.coli.

The Calvin Cycle is the primary conduit for the fixation of carbon dioxide into the biosphere; ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the rate-limiting fixation step. Our goal is to direct the evolution of RuBisCO variants with improved kinetic and biophysical properties. The Calvin Cycle was partially reconstructed in Escherichia coli; the engineered strain requires the Synechococcus PCC6301 RuBisCO for growth in minimal media supplemented with a pentose. We randomly mutated the gene encoding the large subunit of RuBisCO (rbcL), co-expressed the resulting library with the small subunit (rbcS) and the Synechococcus PCC7492 phosphoribulokinase (prkA), and selected hypermorphic variants. The RuBisCO variants that evolved during three rounds of random mutagenesis and selection were over-expressed, and exhibited 5-fold improvement in specific activity relative to the wild-type enzyme. These results demonstrate a new strategy for the artificial selection of RuBisCO and other non-native metabolic enzymes.

Blotting, Western↗

PubMed2003

Enhanced mercury biosorption by bacterial cells with surface-displayed MerR.

The metalloregulatory protein MerR, which exhibits high affinity and selectivity toward mercury, was exploited for the construction of microbial biosorbents specific for mercury removal. Whole-cell sorbents were constructed with MerR genetically engineered onto the surface of Escherichia coli cells by using an ice nucleation protein anchor. The presence of surface-exposed MerR on the engineered strains enabled sixfold-higher Hg(2+) biosorption than that found in the wild-type JM109 cells. Hg(2+) binding via MerR was very specific, with no observable decline even in the presence of 100-fold excess Cd(2+) and Zn(2+). The Hg(2+) binding property of the whole-cell sorbents was also insensitive to different ionic strengths, pHs, and the presence of metal chelators. Since metalloregulatory proteins are currently available for a wide variety of toxic heavy metals, our results suggest that microbial biosorbents overexpressing metalloregulatory proteins may be used similarly for the cleanup of other important heavy metals.

Adsorption↗

PubMed2002

Production of meiosis-activating sterols from metabolically engineered yeast.

Meiosis-activating sterols (MAS), a class of potent regulators of reproductive processes, are difficult to obtain by chemical synthesis or isolation from natural sources. We demonstrate the development of metabolically engineered strains of Saccharomyces cerevisiae that accumulate MAS as the predominant sterol product. Homologous recombination was used to construct an erg24Delta erg25Delta hem1Delta mutant RXY4.3, which lacked sterol Delta14 reductase, C-4 oxidase, and delta-aminolevulinate synthase. The HEM1 deletion allowed sterol import and rendered RXY4.3 viable under aerobic conditions. This mutant accumulated 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS), and a similar erg25Delta hem1Delta mutant produced 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol (T-MAS). Based on consistent yields of approximately 5 mug of FF-MAS per mL of culture, fermentation of genetically modified yeast compares favorably with other approaches to produce MAS.

Cholestadienols↗

PubMed2003

[Screening of Bacillus thuringiensis strains containing vip3A genes and analysis of gene conservation].

Vip3A, a novel insecticidal protein, is secreted by Bacillus thuringiensis (Bt) during vegetative growth. Vip3A protein possesses insecticidal activity against a wild spectrum of lepidopteran insect larvae. Since the first cloning of vip3A gene from Bt, many other vip3A genes have been isolated. To investigate vip3A genes contribution to Bt and reflect the revolution relationships, the strains containing vip3A genes were screened and gene similarity was analyzed. 114 wild-type Bacillus thuringiensis (Bt) strains isolated from different regions and 41 standard Bt strains from the Institute of Pasteur were screened for the vip3A genes using PCR amplification. 39 strains including B. thuringiensis subsp. kurstaki (Btk) HD-1 were found to contain the vip3A genes. Because acrystallerous strain Cry- B derived from Btk HD-1 was proved not to contain vip3A gene, it suppose that the vip3A gene may be located at the plasmids. Vip3A proteins expressed in these strains were detected with polyclonal antibody by Western blot and 4 strains among them were shown not to express the Vip3A proteins. The vip3A genes amplified from wild-type Bacillus thuringiensis strains S101 and 611 with different levels of activity against lepidopteran insect larvae were cloned into pGEM-T Easy vector. Alignment of these 2 putative Vip3A proteins with 6 others (Vip3A (a), Vip3A(b), Vip3A-S, Vip3A-S184, Vip83 and Vip3V) in the GenBank data base and 2 reported Vip3A proteins (Vip14 and Vip15) showed that vip3A genes are highly conservative. The plasmids pOTP-S101 and pOTP-611 were constructed by in- serting 2 vip3A genes (vip3A-S101 and vip3A-611) into the expression vector pQE30 respectively and were transformed into E. coli M15. E. coli M15 cells harboring the pOTP plasmids were induced with 1 mmol/L IPTG to express 89 kDa protein. Experiments showed that the level of soluble proteins of Vip3A-S101 in E. coli M15[pOTP-S101] and Vip3A-611 in E. coli M15 [pOTP-611] were about 48% and 35% respectively. Bioassay showed that each of these Vip3A proteins had similar toxicity against neonate Spodoptera litura larvae, indicating that some amino acids change had little effect on the insecticidal activity of proteins. Although vip3A genes are conservative, the unknown insecticidal spectrum is still to be brought out. Vip3A genes can be used for the construction of the Bt engineered strains and transgenic plants. In addition, vip3A genes are excellent candidates for delay of the pest resistance due to the difference of the action model from that of Bt delta-endotoxins.

Animals↗

PubMed2026

Chemotactic sensing of extracellular antibiotic resistance genes enables their efficient removal by Stutzerimonas stutzeri.

The dissemination of antibiotic resistance genes (ARGs) in wastewater environments poses a severe threat to public health. Extracellular ARGs (eARGs) persist as free DNA fragments that are refractory to efficient removal by conventional physicochemical treatment technologies. Here, we isolated Stutzerimonas stutzeri CHY07 from municipal sewage and demonstrated that extracellular DNA fragments, including eARGs, can serve as chemoattractants for environmental bacteria. Through genomic mining, molecular docking, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and protein-ligand interaction profiling, we identified the chemoreceptor Mcp16 as the primary sensor of extracellular DNA and revealed that it achieves sequence-independent recognition of the DNA phosphate backbone. We further established the endogenous pentapeptide VRSVR as a methylation substrate for CheR and constructed the engineered strain CHY07-2 (mcp16::VRSVR) using an SSB/CRISPR-Cas9 ribonucleoprotein (RNP) system. This strain exhibited significantly enhanced chemotactic responsiveness, achieving 72-h removal efficiencies of 96.56% and 91.60% for low- and high-molecular-weight eARGs in non-sterile WWTP secondary effluent; conversely, mcp16 deletion markedly attenuated both chemotaxis and removal, whereas in situ complementation restored them. These findings reveal a "chemotaxis-contact-removal" cascade - with a proposed self-reinforcing loop - in eARG-removing bacteria, providing both a theoretical framework and a technical paradigm for enhancing pollutant removal through targeted amplification of microbial chemotaxis.

Chemotaxis↗

PubMed1992

Virulence factors of the family Legionellaceae.

Whereas bacteria in the genus Legionella have emerged as relatively frequent causes of pneumonia, the mechanisms underlying their pathogenicity are obscure. The legionellae are facultative intracellular pathogens which multiply within the phagosome of mononuclear phagocytes and are not killed efficiently by polymorphonuclear leukocytes. The functional defects that might permit the intracellular survival of the legionellae have remained an enigma until recently. Phagosome-lysosome fusion is inhibited by a single strain (Philadelphia 1) of Legionella pneumophila serogroup 1, but not by other strains of L. pneumophila or other species. It has been found that following the ingestion of Legionella organisms, the subsequent activation of neutrophils and monocytes in response to both soluble and particulate stimuli is profoundly impaired and the bactericidal activity of these cells is attenuated, suggesting that Legionella bacterial cell-associated factors have an inhibitory effect on phagocyte activation. Two factors elaborated by the legionellae which inhibit phagocyte activation have been described. First, the Legionella (cyto)toxin blocks neutrophil oxidative metabolism in response to various agonists by an unknown mechanism. Second, L. micdadei bacterial cells contain a phosphatase which blocks superoxide anion production by stimulated neutrophils. The Legionella phosphatase disrupts the formation of critical intracellular second messengers in neutrophils. In addition to the toxin and phosphatase, several other moieties that may serve as virulence factors by promoting cell invasion or intracellular survival and multiplication are elaborated by the legionellae. Molecular biological studies show that a cell surface protein named Mip is necessary for the efficient invasion of monocytes. A possible role for a Legionella phospholipase C as a virulence factor is still largely theoretical. L. micdadei contains an unusual protein kinase which catalyzes the phosphorylation of eukaryotic substrates, including phosphatidylinositol and tubulin. Since the phosphorylation of either phosphatidylinositol or tubulin might compromise phagocyte activation and bactericidal functions, this enzyme may well be a virulence factor. Administration of the L. pneumophila exoprotease induces lesions resembling those of Legionella pneumonia and kills guinea pigs, suggesting that this protein plays a role in the pathogenesis of legionellosis. However, recent work with a genetically engineered strain has convincingly shown that the protease is not necessary for intracellular survival or virulence. As might be expected with a complex process like intracellular parasitism, it appears that the capability of Legionella strains to invade and multiply in host phagocytes is multifactorial and that no single moiety which is responsible for the virulence phenotype will be found.

Humans↗

PubMed1998

The contribution of caseins to the amino acid supply for Lactococcus lactis depends on the type of cell envelope proteinase.

The ability of caseins to fulfill the amino acid requirements of Lactococcus lactis for growth was studied as a function of the type of cell envelope proteinase (PI versus PIII type). Two genetically engineered strains of L. lactis that differed only in the type of proteinase were grown in chemically defined media containing alphas1-, beta-, and kappa-caseins (alone or in combination) as the sources of amino acids. Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase. Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation. This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for L. lactis in milk (S. Helinck, J. Richard, and V. Juillard, Appl. Environ. Microbiol. 63:2124-2130, 1997) as a function of the type of proteinase. Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined. The results were in agreement only in the case of the PI-type proteinase. Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a PIII-type proteinase.

Amino Acid Sequence↗

PubMed2001

Genetically engineered Pseudomonas: a factory of new bioplastics with broad applications.

New bioplastics containing aromatic or mixtures of aliphatic and aromatic monomers have been obtained using genetically engineered strains of Pseudomonas putida. The mutation (-) or deletion (Delta) of some of the genes involved in the beta-oxidation pathway (fadA(-), fadB(-) Delta fadA or Delta fad BA mutants) elicits a strong intracellular accumulation of unusual homo- or co-polymers that dramatically alter the morphology of these bacteria, as more than 90% of the cytoplasm is occupied by these macromolecules. The introduction of a blockade in the beta-oxidation pathway, or in other related catabolic routes, has allowed the synthesis of polymers other than those accumulated in the wild type (with regard to both monomer size and relative percentage), the accumulation of certain intermediates that are rapidly catabolized in the wild type and the accumulation in the culture broths of end catabolites that, as in the case of phenylacetic acid, phenylbutyric acid, trans-cinnamic acid or their derivatives, have important medical or pharmaceutical applications (antitumoral, analgesic, radiopotentiators, chemopreventive or antihelmintic). Furthermore, using one of these polyesters (poly 3-hydroxy-6-phenylhexanoate), we obtained polymeric microspheres that could be used as drug vehicles.

Biodegradation, Environmental↗

PubMed2000

Assessment of the carcinogenicity associated with oral exposures to hydrogen peroxide.

Concern regarding hydrogen peroxide (H(2)O(2)) carcinogenicity arises from its ability to act as a strong oxidizing agent. In short-term genotoxicity tests, H(2)O(2) has given predominantly positive results; however, these assays have been performed using either bacterial strains engineered to be exquisitely sensitive to oxidant damage, or mammalian cells deficient in antioxidant enzymes. Significantly, the addition of antioxidant protective measures (normally present in vivo) to these assay systems protects against H(2)O(2) genotoxicity. In most whole animal studies, H(2)O(2) exposure neither initiates nor promotes tumors. In mice, however, 0.4% H(2)O(2) in drinking water was reported to induce hyperplastic lesions of the duodenum and to erode areas in the glandular stomach epithelium. Owing to the chemistry of dilute H(2)O(2) solutions and the anatomy/physiology of the gastrointestinal tract, it is unlikely that orally ingested H(2)O(2) reaches the duodenum. Instead, greatly decreased water consumption and the resultant abrasion of the luminal lining on ingestion of pelleted dry rodent chow is the most likely cause of the observed gastric and duodenal lesions following H(2)O(2) administration in drinking water. Significantly, when hamsters received high doses of H(2)O(2) by gastric intubation (and water intake was not affected), the gastric and duodenal epithelia appeared normal. In-depth analysis of the available data supports the conclusion that oral ingestion of H(2)O(2) should not be considered a carcinogenic threat.

Administration, Oral↗