An automatic,on-line glucose analyzer for feed-back control of fed-batch growth of Escherichia coli

Summary A computer-assisted on-line glucose analyzer was developed for feed-back control of cell growth. Using this system the glucose consumption rate for Escherichia coli was determined to be linear during batch culture at 0.37 g/hr. On-line feed-back control of glucose concentration at 1.5±0.5 g/L was used with fed-batch cultures to produce 31.2 g dry weight of E. coli cells/L in 12 h.     

Continuous culture of a recombinantEscherichia coli and plasmid maintenance for tryptophan production

Summary Production of tryptophan by a temperature sensitive recombinant microorganism (Escherichia coli W3110 trpLDtrpR ^ts tna ^– (pCRT185)) was investigated. In a single-stage continous culture, at an elevated temperature, 42°C (derepressed condition), tryptophan concentration increased in an early phase of the fermentation, and then gradually decreased with time. The reduction in the production rate was mostly due to the segregation of the plasmid and subsequent increase of plasmid-free cells. However, the plasmid could be maintained stable at 37°C, with repressed condition oftrp-operon, over 200 generations. A two-stage continuous culture system, i.e. cell growth was maintained in the first stage at 37°C and gene expression was induced in the second stage at 42°C, was therefore tested to improve the performance of the fermentation system. Operation of the two-stage system showed that the plasmid stability was significantly improved, and the specific rate of tryptophan production was maintained almost constant for more than 500 hours in the second stage.     

Production of cloned carboxypeptidase G2 by Escherichia coli: Genetic and environmental considerations

Summary The optimum production of cloned carboxypeptidase G₂ from plasmid pNM21 byEscherichia coli was found to be strongly strain- and temperature-dependent. The superior host was strain RV308 and the preferred growth temperature 28°C. Copy number, which decreased during exponential growth of all strains examined, did not relate in these studies to the level of enzyme production: the strain with the highest enzyme yield also having the lowest overall copy number.     

Utilization ofEscherichia coli tac promoter inBacillus subtilis

Summary The tac promoter originally constructed for the use inEscherichia coli was fused to an endoglucanase structural gene isolated fromBacillus subtilis and the expression of the chimeric gene inB. subtilis was observed. The tac promoter-controlled gene expressed well inB. subtilis and produced endoglucanase during the exponential growth phase.     

Overproduction of human insulin-like growth factor-II inEscherichia coli

Summary Human insulin-like growth factor II (hIGF-II) was produced inEscherichia coli as a protein fused to human growth hormone. High level expression of the fusion protein was attained with pIBL-1 plasmid. The hIGF-II obtained byin vitro cleavage of the fusion protein with cyanogen bromide was highly purified and its biological activity was assessed.     

Analysis of the ArcA regulon in anaerobically grown <Emphasis Type="Italic">Salmonella enterica sv</Emphasis>. Typhimurium

Background  

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative pathogen that must successfully adapt to the broad fluctuations in the concentration of dissolved dioxygen encountered in the host. In Escherichia coli, ArcA (Aerobic Respiratory Control) helps the cells to sense and respond to the presence of dioxygen. The global role of ArcA in E. coli is well characterized; however, little is known about its role in anaerobically grown S. Typhimurium.     

Accumulation of cyclic guanosine 3':5'-monophosphate in the culture medium of growing cells of Escherichia coli.

In an exponentially growing culture of E. coli, the concentration of cyclic guanosine 3′:5′-monophosphate (cyclic GMP) was found to increase in parallel with the bacterial growth. As the cells approach the stationary phase of growth, the increment of cyclic GMP also ceases progressively to reach to a plateau. When cells are separated from the medium by centrifugation, almost all of the cyclic GMP is recovered in the culture supernatant. The amount of cyclic GMP accumulated is proportional to the number of cells present in the culture. These results suggest that a constant number of cyclic GMP molecules is synthesized each generation of E. coli, and is excreted from the cells to accumulate into the medium.     

Improved L-lysine production by the amplification of theCorynebacterium glutamicum dapA gene encoding dihydrodipicolinate synthetase inE. coli

Summary ThedapA gene (L-2,3-dihydrodipicolinate synthetase: DHDP synthetase) ofCorynebacterium glutamicum JS231, a lysine overproducer, cloned and subcloned inE. coli/C. glutamicum shuttle vector pECCG117 was used to transformE. coli threonine producer and threonine and lysine coproducer. The plasmid pDHDP5812 carryingdapA gene ofC. glutamicum led to increase in lysine production in theseE. coli strains. Threonine and lysine co-producerE. coli TF1 with pDHDP5812 produced lysine with small amount of threonine. The DHDP synthetase activity ofE. coli TF1 carrying pDHDP5812 showed high resistance toward inhibition by lysine.     

A high molecular weight plasmid ofZymomonas mobilis harbours genes for HgCl2 resistance

Summary Plasmids fromZ. mobilis could be stably maintained inE. coli HB101 in which the expression of various drug resistance markers could be monitored. A large molecular weight plasmid (5.2 kbp) ofZ. mobilis was found to harbour the genes for mercuric chloride degradation and to confer uponE. coli, resistance to a higher mercuric chloride concentration as compared toZ. mobilis. The introduction of this plamsid madeE. coli sensitive to concentrations of cadmium acetate which were originally non-inhibitory to it.     

Glyoxalase-I activity and cell cycle regulation in yeast

The status of glyoxalase-I was explored in exponentially growing and G₁ arrested temperature sensitive (ts) cell division cycle (cdc) mutants of Saccharomyces cerevisiae. It was observed that the specific activity of this enzyme was correlated with overall growth status. The activity was high in actively growing cells and was low in G₁ arrested cells. Specific activities of glyoxalase-I were also low in G₁ arrested prolonged stationary phase (PSP) cells of S. cerevisiae and Candida albicans. The activity of glyoxalase-I recovered when G₁ arrested S. cerevisiae (ts) cells were allowed to regrow under permissive conditions. Results demonstrate that although glyoxalase-I activity is a good indicator of cell growth status, it is not involved in cell cycle regulation of this eukaryotic organism.     

Specific differences in tolerance to exogenous berberine among plant cell cultures

The tolerance of plant cells to exogenously administered berberine, an antimicrobial isoquinoline alkaloid, was studied using berberine-producing and nonproducing cell suspension cultures. Both Coptis japonica and Thalictrum flavum cells, which have an intrinsic ability to synthesize berberine, took up exogenous berberine from the culture medium by an energy-requiring active transport to accumulate it exclusively in vacuoles. By contrast, T. minus cells, which excrete indigenous berberine mostly into the medium, did not take up exogenously supplied berberine, indicating that the alkaloid transport in this species is unidirectional. No inhibition of cell growth by exogenous berberine was observed in the three berberine-producing cell cultures. On the other hand, a small amount of exogenous berberine strongly inhibited cell growth in the berberine-free cultures of Datura innoxia, Catharanthus roseus, and Paeonia albiflora. The berberine taken up actively by Datura cells could not be transported into vacuoles but was dispersed in the cytoplasm, causing a severe inhibition of cell growth.     

Physiological constraints in increasing biomass concentration ofEscherichia coli B in fed-batch culture

Summary Fed-batch culture ofE.coli B was carried out to obtain high concentration of biomass. After requirement of oxygen was met by sparging the pure oxygen, physiological constraints were delineated. High partial pressure of CO₂ caused the decrease of the maximum specific growth rate, whereas fermentative byproducts caused the decrease of biomass yield as well as the maximum specific growth rate.     

Production of active human interferon-β inE. coli II. Optimal condition for induced synthesis by 3,β-indoleacrylic acid

Summary The maximum level of human interferon- activity was expressed under the control of theE. coli tryptophan promoter whenE. coli cells were induced at late logarithmic growth phase by 3,-indoleacrylic acid (IAA). The level is one order of magnitude higher than that obtained when the cells were induced at early logarithmic or stationary phase. When IAA was subsequently further added, the decrease in the activity observed at a latter period of fermentation was suppressed.     

Thermostable tryptophan synthase ofBacillus stearothermophilus expressed inEscherichia coli

Summary The tryptophan synthase genes,trpA andtrpB, from a moderate thermophile,Bacillus stearothermophilus IFO13737, were expressed efficiently inEscherichia coli. The recombinant tryptophan synthase amounted to 22% of the soluble cellular protein, and was purified to homogeneity by three steps. The enzyme is more thermostable thanE.coli tryptophan synthase, especially the subunit. The enzyme is also more resistant to sodium dodecylsulfate and methanol thanE.coli enzyme.     

Resistance of bacterial film to contamination during use as inoculum in repeated batch fermentation

Summary AnEscherichia coli strain constitutive for -galactosidase was immobilized onto cotton cloth. The resultingE.coli film was used as a resident inoculum in repeated batch fermentations for 30 days in the presence ofBrevibacterium ammoniagenes added as a contaminant. Analysis of -galactosidase production shows that contamination did not decrease the capacity of the film to generateE.coli cells, or decrease theE.coli population on the film.     

Higher culture pH is preferable for inclusion body formation of recombinant salmon growth hormone inEsherichia coli

Summary Higher culture pH of 7.6 was shown to be preferable for the inclusion body formation of salmon growth hormone (SGH) inEscherichia coli harboring a recombinant plasmid. High-level formation of SGH inclusion bodies was achieved at 33°C (pH 7.6). Growth inhibition by soluble SGH was also observed.     

Elicitation of anthocyanin production in cell cultures of carrot (Daucus carota L) by using elicitors and abiotic stress

Summary A cell line of carrot (Daucus carota L) which produces anthocyanin was subjected to various elicitors and abiotic stresses: The elicitors tested were culture filtrates (CF) and cell extracts (CE) of certain bacteria and yeasts. The abiotic stresses were salts of certain metal ions. The production increase obtained with cell extracts of Bacillus cereus. Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus were 49, 72, 45 and 41% respectively over the control. Maximum elicitation was obtained with elicitor derived from cell extract of the yeast Rhodotorula rubra where it enhanced anthocyanin production by two fold. The abiotic stress agents Ca, Mn, Zn, Co, Fe & V enhanced anthocyanin production. Of all the metal ions tested Ca was the most effective. The elicitation process was governed by the type and level of elicitor.     

Purification of recombinant salmon growth hormone expressed inEscherichia coli

Summary Recombinant salmon growth hormone (SGH) expressed inEscherichia coli was refolded and purified. Native form SGH with a purity of 98% was obtained with a recovery of 9%. We found that purified SGH in reduced form under denaturing conditions efficiently formed correct disulfide bonds.     

The effect of the H2 partial pressure on the metabolite pattern ofLactobacillus casei,Escherichia coli andClostridium butyricum

Summary The metabolite pattern of batch cultures ofLactobacillus casei LMG 6400,Clostridium butyricum LMG 1213t1 andEscherichia coli LMG 2093 was effected only for the latter organism when the H₂ partial pressure was below 1 atmosphere: high hydrogen partial pressures increased the formate formation, low pressures gave rise to increased acetate production and higher cell yields.     

Construction of aClostridium acetobutylicum-Escherichia coli shuttle vector

Summary A 4.1-kb cryptic plasmid, designated pCA134, has been isolated fromClostridium species. In order to develop a vector suitable for transforming saccharolytic clostridia three hybrid plasmids were constructed by inserting pCA134 into pHV32 withEcoRI, orBglII andBamHI. The newly constructed plasmids were propagated inEscherichia coli and were used to transformBacillus subtilis andClostridium acetobutylicum. One of them, pCAB32 (10.1 kb), which contains chloramphenicol acetyltransferase gene and an origin of replication derived from pCA134 was introduced intoB.subtilis andC.acetobutylicum as well asE.coli.