Expression of Escherichia coli tryptophan operon in Rhizobium leguminosarum.

Summary RP4-trp hybrid plasmid containing Escherichia coli whole tryptophan operon was conjugatively transferred from E. coli to Rhizobium leguminosarum strains carrying mutations in different trp genes, converting their Trp^– phenotype to Trp⁺. That the phenotype change of the R. leguminosarum cells was due to the presence of the E. coli tryptophan operon was verified by the isolation of RP4-trp hybrid plasmid from the R. leguminosarum conjugant cells, and by re-transfer of RP4-trp plasmid by conjugation back to E. coli trp and Pseudomonas putida trp strains. Enzymatic activities of anthranilate synthetase and subunit of tryptophan synthetase in crude extracts of R. leguminosarum cells containing RP4-trp plasmid were much higher than that of the wild-type cells and were not repressed by the presence of tryptophan in the culture medium.     

Production of active human interferon-β inE. coli I. Preferential production by lower culture temperature

Summary Unusually low culture temperature, such as 20°C, was shown to be preferable for the synthesis of active human interferon- (IFN-) inE. coli harboring a recombinant plasmid. TheE. coli cells cultured at 20°C gave 8.6-fold higher IFN- activity than those cultured at 37°C. However, almost the equal amounts of IFN- protein were accumulated in both cells cultured at 20°C and at temperature higher than 20°C, suggesting that IFN- might exist as an active form in the cells cultured at 20°C, while as a rather denatured form in the cells cultured at higher temperature.     

Temperature optimization ofin vivo expression from theE. coli trp andtrp::lac promoters

Summary We have studied the effect of temperatures between 20°C and 40°C on the activity ofE. coli promoters. TheE. coli lac UV5,trp::lac, andtrp promoters reach their maximal activity at 37°C while thetet::trp promoter reaches its maximal activity at 40°C. The addition of 5g IAA/ml was sufficient to derepress thetrp promoter over the temperature range studied.     

Release of β-galactosidase from E. coli by a plasmid containing a temperature sensitive lytic function

Summary We report the construction of a plasmid which carries the øX174 lysis gene E downstream from the lambda pL promoter. This plasmid is capable of triggering lysis inE. coli when the growth temperature is raised from 30°C to 42°C. The kinetics of release of -galactosidase and intracellular protein have been determined, as well as the decrease in cell viability upon induction of lysis. In addition, -galactosidase can be released efficiently after induction by a short sonication period. Both methods allow efficient release of -galactosidase.     

Enhancement of tryptophan production by Escherichia coli as an application of genetic engineering

Summary A feedback resistant trp operon plasmid that transformed a multiple mutant (trpR tnaA) of Escherichia coli was found to enhance remarkably the production of tryptophan in a bench-scale fermentor. 5.5 g of tryptophan was accumulated per litre of culture medium at 24th hr in batch. The productivity was 0.229 g/l/hr. This productivity is the highest among those ever reported by other workers. The recombinant plasmid (Tc^r Trp⁺ I^-) used was completely stable in each run when tetracycline was added by 10 g/l into the medium.     

Effect of temperature on plasmid stability and expression of cloned cellulase gene in a recombinantBacillus megaterium

Summary The expression of carboxymethyl cellulase gene inBacillus megaterium (pCK108) was investigated with respect to temperature in batch culture. The suboptimal temperature supporting maximal cell growth rate was determined to be 30 °C at which stability of the plasmid pCK108 could be maintained stable. However, cellular plasmid contents, production rate of cellulase of the cell, and efficiency of the gene expression increased significantly with increase of the temperature from 30 °C to 44 °C, even though the plasmid stability decreased up to 60% level at the end of the culture.     

Cloning a mutatedtrp operon for the biosynthesis of an antibiotic agent

Summary Plasmid pCG8 containing a mutatedtrp operon was constructed and introduced into anEscherichia coli host for increasing tryptophan biosynthesis. Since tryptophan is a biosynthetic precursor of pyrrolnitrin, the mutated trp operon was inserted into aPseusdomonas vector pME290 to obtain the plasmid pCG12, and then was introduced intoPseudomonas pyrrocinia for enhancing the pyrrolnitrin synthesis. Data showed that the production of pyrrolnitrin of the transformed strain was five-times greater than that of the parental strain.     

Induction of freezing tolerance in microspore-derived embryos of winter Brassica napus

Freezing tolerance was induced in microspore derived embryos of winter Brassica napus cv. Jet neuf by the addition of ABA or mefluidide to the culture media during embryogenesis. Survival after freezing was estimated by culture of frozen-thawed embryos to plantlets. A higher freezing tolerance (50% survival at –15°C) was induced when 50 M ABA or 3.2 M mefluidide was incorporated initially into the medium during embryogenesis at 25°C followed by culture at 2°C for 3 weeks. When embryos were induced in the absence of ABA or mefluidide and maintained at 2°C for even as long as 12 weeks a lower degree of freezing tolerance (10% survival at –15°C) was obtained. Plants regenerated from embryos hardened maximally by a combination of either ABA or MFD with low temperature did not require further vernalization for flowering.Abbreviations ABA abscisic acid - MFD mefluidide - 2,4-D 2,4-dichlorophenoxyacetic acid - LT₅₀ killing temperature for 50% of the embryos     

Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min^–1 mg^–1, respectively.     

A new runaway type episomal vector for mammalian cells based on a temperature-sensitive simian virus 40 and inducible erythropoietin production

A runaway vector for mammalian cells was constructed from the simian virus 40 (SV40) genome with a temperature-sensitive mutation of the large T antigen and bacterial neo ^r gene. Replication of this plasmid was repressed above 39°C and vigorous DNA propagation was observed below 33°C in simian CV-1 cells. The human erythropoietin gene was inserted downstream of the SV40 late promoter of the plasmid and the recombinant plasmid was introduced into CV-1 cells. By a temperature shift from 37 to 33°C, the plasmid copy number increased from 5 × 10² to 5 × 10³ copies per cell and the specific production rate of erythropoietin increased more than ten-fold. The bacterial-derived sequences such as the neo ^r gene and vector pUC sequences were prone to delete but the main body of the recombinant plasmid such as SV40 and the erythropoietin-coding sequences were stably maintained at either 33 or 37°C.     

Ethanol production from glucose byClostridium thermosaccharolyticum strains: Effect of pH and temperature

Summary The fermentation of glucose byClostridium thermosaccharolyticum strains IMG 2811^T, 6544 and 6564 was studied in batch culture in a complex medium at different temperatures in defined and free-floating pH conditions. All the strains ferment 5 g glucose.l^–1 completely. The yield of the fermentation products turned out to be independent of the incubation temperature for strain IMG 2811^T. Strain IMG 6544 produced at 60°C significantly more ethanol and less acetic acid, butyric acid, hydrogen gas and biomass than at lower temperatures. With strain IMG 6564, the opposite effect occurred: ethanol appeared to be the main fermentation product at 45°C; at 60°C less ethanol and more acetic acid, butyric acid and hydrogen gas was formed.Experiments, carried out with strain IMG 6564, at defined pH conditions (between 5.5 and 7) and different temperatures (45, 55 and 60°C) revealed no effect of the incubation temperature, but an important effect of the pH on the product formation. At pH 7, ethanol was the main fermentation product while minor amounts of hydrogen gas, acetic and butyric acid were produced. Lowering the pH gradually to 5.5 resulted in a decrease of ethanol and an increase of biomass, hydrogen gas, acetic, butyric and lactic acids. At pH higher than 7 no growth occurred. Similar conclusions could be drawn for strains IMG 2811^T and 6544.     

A new way of stabilizing recombinant plasmids

A new method to stabilize recombinant plasmids extremely well was exploited using Escherichia coli Tna (trpAEI trpR tnaA) and pSC101trpI15-14 (tetracycline resistance, whole trp operon) as a model system. We mutagenized the Tna strain carrying pSC101trpI15-14 and isolated a mutant 6F484 that stably maintained the recombinant plasmid for 100 generations. From 6F484, plasmid-free cells (tetracycline sensitive) were screened for on selective agar plates containing fusaric acid. The host strain FA14 was found to have lost the ability for active transport of tryptophan, in addition to the phenotype of Trp⁻. Therefore, strain FA14 could not grow normally even in a complete medium. However, when the strain was transformed with the trp operon recombinant plasmid, its growth rate was almost restored to the original level. These results suggest that the recombinant plasmid is indispensable for the normal growth of host cells like FA14. Even if plasmid-free segregants appear during the cultivation, they cannot grow so rapidly and are diluted as a minority in total population. Consequently, owing to the deficiency of both the biosynthesis and uptake of tryptophan in host strain, the trp operun recombinant plasmid can be stably maintained.     

Solid state fermentation of straw withNeurospora crassa for CMCase and β-glucosidase production

Summary CMCase and -glucosidase were produced by the mutantNeurospora crassa 40b cultivated on untreated wheat straw in a solid state fermentation. Best enzyme activities were observed when the growth medium was composed of wheat straw mixed with certain mineral solutions at a ratio 1:2 (w/v). A partially purified enzyme preparation showed optimum enzyme activities of CMCase and -glucosidase at pH 4.0 and 5.0 and temperature 50 and 60°C respectively. The apparent Km values for the same enzymes were 16.8 g/l and 1.03x10^–4 M respectively. At optimum growth and enzyme assay conditions yields as high as 586.2 U CMCase and 58.4 U -glucosidase per gram of straw were obtained.     

Purification and characterisation of the extracellular maltase ofPaecilomyces varioti CMI 16196

Summary The extracellular maltase of the fungusPaecilomyces varioti CMI 16196 was purified by acetone precipitation and Sephadex G-150 gel filtration. The enzyme functioned optimally at pH 3.5–4.0 and 60°C. The maltase hydrolysed maltose and maltotriose preferentially but was inactive on both the aryl-and alkyl--D-glucosides. The Km for maltose was 1.31 mM. End product configuration was maintained and transferase activity was observed.     

Development of a fermentation procedure to produce a tempe-related food using common beans as substrate

Summary A basic procedure was developed to produce a tempe-like product using the mouldRhizopus oligosporus and black common beans (Phaseolus vulgaris) as substrate. The initial pH of the substrate was 5.8, and fermentation was conducted at 37°C with a relative humidity of 70% for 72 hrs. Levels of soluble solids and soluble protein increased dramatically as a result of fermentation. Some changes were as well observed in fatty acid contents of fermented samples. It was concluded that the common bean used was an acceptable substrate for preparing this product.     

Isolation of aClostridium thermocellum gene encoding a thermostable β-1, 3-glucanase (laminarinase)

Summary AClostridium thermocellum gene directing the synthesis of a thermostable -glucanase was localized on a 1.9-kb DNA fragment by subcloning intoEscherichia coli plasmid vectors. The enzyme was highly efficient in degrading glucans with alternating -1, 3- and -1,4-linkages such as lichenan and barley glucan. It was also active towards the -1, 3-glucan laminarin, but lacked activity on cellulosic substrates and -glucans. The enzyme was therefore classified as -1, 3-glucanase (laminarinase) and the corresponding gene was designatedlicA. With barley -glucan as substrate the enzyme had a pH optimum around pH 6.5 and a temperature optimum at 65°C. It was stable for several hours at 60°C in the absence of substrate.     

Production of ethanol at high temperatures in the fermentation of Jerusalem artichoke juice and a simple medium byKluyveromyces marxianus

Summary Temperatures as high as 36°C and 40°C did not negatively affect the ethanol productivity of Jerusalem artichoke (J.a.) juice batch fermentation and the final concentrations of ethanol were close to those produced at lower temperatures. At higher process temperatures (36–40°C), ethanol toxicity inKluyveromyces marxianus was less important during the fermentation of J.a. juice as compared with a simple medium. In simple medium, the heat-sticking of fermentation was observed and the percentage of unfermented sugars steeply increased from 28°C up to 40°C.     

Plasmid pKM101-mediated mutagenesis in Escherichia coli is inducible

Summary A tif-1 umuC36 double mutant of Escherichia coli was constructed. It has been found that the umuC36 mutation prevents both increased spontaneous mutagenesis and enhanced reactivation of UV-irradiated , phenomena normally observed in the tif-1 strain grown at 42°C. When the plasmid pKM101 was introduced into tif-1 umuC36, an elevated spontaneous reversion rate of the his-4 mutation observed at 30°C was further increased 6-fold at 42°C. This was accompanied by a 10-fold increase in the ability of tif-1umuC36 containing pKM101 and grown before infection at 42°C to reactivate UV-irradiated .     

Thermo-osmoregulation of Heat-Labile Enterotoxin Expression by <Emphasis Type="Italic">Escherichia coli</Emphasis>

Abstract:Enterotoxigenic Escherichia coli causes diarrhea by producing several virulence factors including heat-labile enterotoxin (LT). LT is maximally expressed at 37°C. The histone-like nucleoid structuring protein (H-NS) appears to inhibit LT expression by binding to a downstream regulatory element (DRE) at low temperatures. An hns⁺ E. coli strain, X7026, carrying an LT–beta-galactosidase translational fusion plasmid (pLT-lac) was shown to be responsive to varying amounts of sodium chloride (NaCl) as well as sucrose or lithium chloride. Maximal responsiveness to the various osmolytes was obtained with cells grown at 37°C under microaerophilic conditions. Temperature-osmotic upshift experiments demonstrate LT expression is thermo-osmoregulated. pLT-lac was tested in an hns strain or its congenic hns⁺ strain for its response to NaCl. LT expression is elevated in the hns strain regardless of NaCl concentration and retains its osmoresponsiveness. The response of the DRE deletion plasmid (pLT-lacNC) to NaCl is similar to that of the undeleted plasmid.     

Spectrophotometric titrations of ferricytochrome C with ferrohexacyanide in the pH range 5 to 7

Spectrophotometric titrations were conducted on the system horse heart ferricytochromec plus ferrohexacyanide in the pH range 5 to 7 and at temperatures 8, 18, 22 and 28°C. A difference extinction coefficient for reducedvs. oxidized cytochromec at 550 nm of 21 mmol^–1cm^–1 was used in part of the evaluations. On the assumption that only one electron-transferlinked proton dissociation is effective for both ferro- and ferricytochromec in this pH range, various possible models are developed with only three conforming with the experimental pH dependence of the spectrophotometric equilibrium constant. The data conform best to a model with protonic dissociation constants between pH 5 and 7 such that the reduced cytochromec species is at least a factor of 3 more acidic than the one for oxidized cytochromec (with pK_H 6). This interpretation holds least for the data at 22°C, which points to a structural rearrangement at about this temperature (Czerlinski and Bracokova, 1973; Zabinski and Czerlinski, 1974; Zabinski, et al., 1974). While the extinction coefficient of ferrocytochromec shows no significant change with pH and temperature, the one for ferricytochromec does: it is about 5% larger at pH 5 than at pH 7 (550 nm). Graphs for the absorption change of ferricytochromec (pH 7 as reference) document the details over the wavelength range 500 to 750 nm.