A new control strategy for yeast production based on the L/A* approach

Summary We studied the effect of temperature on the production of an extracellular neutral metalloproteinase of Bacillus megaterium in a laboratory fermentor under constant aeration and pH. The optimal temperature for growth (35–38° C) was higher than that for the synthesis of proteinase during exponential growth (below 31° C). The critical biomass concentration at which the exponential growth terminated decreased with increase in cultivation temperature. The specific rate of proteinase synthesis decreased when the critical biomass concentration was achieved. The observed decrease in proteinase synthesis was related to the cultivation temperature. The temperature also influenced the level of mRNA coding for proteinase. We formulated a mathematical model of cultivation describing the dependence of growth and proteinase synthesis on dissolved oxygen and temperature. The parameters of the model were identified for temperature intervals from 21 to 41° C using a computer. The optimum temperature for the enzyme production was 21° C. The productivity (enzyme activity/time) was maximal at 24–28° C. When optimizing the temperature profile of cultivation, we designed a suboptimal solution represented by a linear temperature profile. We have found that under conditions of continuous decrease in temperature, the maximal production of the proteinase was achieved at a broad range of temperature (26–34° C) when the rate of temperature decrease was 0.2–0.8° C/h. The initial optimal temperature for the enzyme productivity was in the range of 32–34° C. The optimum temperature decrease was 0.8° C/h. Offprint requests to: J. Chaloupka     

Characterization of an extracellular glycoprotein inhibitor of trypsin (TI-23) produced by Streptomyces sp. 23

Summary The production of an extracellular trypsin inhibitor, TI-23, was found to parallel the growth of Streptomyces sp. 23 at different cultivation temperatures, reaching a maximum level at late exponential phase. Although the different temperatures (18°, 28° and 37°C) did not greatly affect the growth of the microorganism, they proved to be an important factor for extracellular inhibitory activity. Maximum specific rates of both cell growth and production of the inhibitor were recorded during the cultivation of Streptomyces sp. 23 at 37°C. TI-23 proved to be a monomeric glycoprotein containing 17% carbohydrate and differing in amino acid composition from the known extracellular proteinase inhibitors of streptomycetes. The molecular mass of the inhibitor was estimated to be about 13 kDa and the isoelectric point 4.3. The inhibition spectrum of TI-23 included trypsin as well as some microbial alkaline proteinases.     

Temperature-sensitive initiation of DNA replication in a mutant of Escherichia coli K12

Summary A mutant of E. coli K12 appears to be temperature-sensitive in the process of initiation of DNA replication. After a temperature shift from 33 to 42°C, the amount of residual DNA synthesis (Fig. 1) and the number of residual cell divisions (Figs. 2,4) indicate that rounds of DNA replication in process are completed, but new rounds cannot be initiated. Following the alignment of chromosomal DNA by amino acid starvation at 33° C no residual DNA synthesis at 42°C takes place (Fig. 5). When the temperature is lowered to 33°C after a period of inhibition at 42°C, the following observations are made: 1. DNA replication resumes and proceeds synchroneously, (Figs. 7, 8a), 2. cells start to divide again only after a lag period of about 1 hour 3. a temporary increase in cell volume is correlated with the frequency of initiation of DNA synthesis (Fig. 8a, b). In a lysogenic mutant strain prophage is inducible; with all bacteriophages tested, replication of phage DNA is not inhibited at 42°C.     

The heat shock response inLocusta migratoria

Summary Locusta migratoria adults reared at 27–30°C die after 2 h at 50°C, but they survive this temperature stress if first exposed to 45°C for 0.5 to 4.5 h. Fat bodies from adult females produce a set of at least six specific polypeptides with molecular weights of 81, 73, 68, 42, 28, and 24×10³ in reponse to heat shock (39–47°C for 1.5 h). These molecular weights closely match those of the heat shock proteins (hsps) observed inDrosophila, with the possible exception of the 42 kd protein of locusts. The optimal temperature for induction of hsps in locusts is 45°C, which is one of the highest heat shock temperatures reported in metazoans. The correspondence between the optimal temperature for hsp induction and the temperature at which enhanced heat tolerance is acquired (both 45 °C) suggests that the hsps may be associated with thermal protection in these insects.There appears to be no substantial translational control in the locust heat shock response, since other proteins are produced, albeit with some reduction, under heat shock conditions. Vitellogenin synthesis in fat bodies at 45°C is 55% of that observed at 30°C. The high optimal heat shock induction temperature and the continued synthesis of non-heat shock proteins may be adaptive to the locust's natural environment.     

Effect of temperature on translation of mRNA coding for an extracellular proteinase and cell proteins inBacillus megaterium

The content and the half-life of mRNA coding for the Ca²⁺-dependent metalloproteinase were measured by determining the enzyme activity excreted into the medium by cells pregrown in the absence of Ca²⁺ after addition of Ca²⁺ and actinomycin D. The content of the functional proteinase mRNA was highest at 31°C, which is the optimal temperature for the synthesis of this enzyme. Its half-life was 15 min, 7 min, and less than 2 min at 24°, 35°, and 42°C, respectively. Only the third of mRNA molecules synthesized at 31°C was translated in vivo into an active enzyme at 42°C, when compared with the translation proceeding at 24°C. Two-thirds of mRNA molecules synthesized at 31°C were translated into stable cell proteins at 42°C when compared with translation at 24°C. The mean half-life of mRNAs coding for cell proteins was 6–7 min at 24°C, 3 min at 35°C and 2 min at 42°C.     

Heat and cold shock responses in different strains of Thiobacillus ferrooxidans

Different strains of Thiobacillus ferrooxidans were examined for their ability to produce a heat shock and a cold shock response. Strain A1, heat shocked from 20° to 35°C, acquired thermotolerance, as it showed a 1000-fold reduction in cell mortality when exposed to the supermaximum temperature of 42°C, as compared to a non-heat-shocked control. A heat shock from 25° to 35°C yielded similar results, although a higher degree of thermotolerance was achieved for the shorter exposure times. Cultures heat shocked for 5 h showed a five-log reduction in viable counts after 41 h at 42°C, whereas non-heat-shocked cultures showed a similar reduction in viability in 28 h. Conferred thermotolerance was immediate and sustained for the duration of the exposure to 42°C. Heat-shocked cultures were not significantly protected against loss of viability due to freezing (-15°C for 24 h). Strain S2, cold shocked from 25° to 10°C, and strain D6, cold shocked from 25° to 5°C, were not protected against freezing at-15°C. An analysis of proteins extracted from heat-shocked cells of strain A1 showed the presence of at least one newly induced protein and eight hyper-induced proteins. The molecular weights of the heat shock proteins were in the range of 15–80.3 kDa.     

Heat sensitivity and protein synthesis during heat-shock in the tobacco hornworm,Manduca sexta

Summary Fifth instar larvae of the tobacco hornworm,Manduca sexta, tolerate 1-h exposures to temperatures as high as 42°C. Above 42°C, survival declines rapidly to 18% at 44°C and 0% at 48°C. As in other insects, the heat-shock response ofManduca sexta involves the induction of synthesis of heat-shock proteins very similar in size to theDrosophila heat-shock proteins (84, 73, 71, 27, 25, 23, and 22 kd). In the epidermis, heat-shock protein synthesis peaks at 42°C, correlating with the heat sensitivity of both the tissue itself and the intact larva. Some heat-shock proteins have different isoelectric forms depending on tissue. Also, the heat-shock proteins are synthesized over a wider range of temperatures in the imaginal discs and the fat body as compared to the epidermis. In contrast to dipteran insects,Manduca sexta does not exhibit a strong repression of non-heat-shock protein synthesis under tolerable conditions.Abbreviations TCA trichloroacetic acid - PAGE polyacrylamide gel electrophoresis - AZT arbitrary Zeitgeber time - kd kilodaltons     

Temperature sensitive initiation of DNA synthesis in a mutant of Salmonella typhimurium

Summary A mutant (dna-1) of Salmonella typhimurium defective in DNA synthesis is described. DNA synthesis is stopped in this mutant at 42° after a residual synthesis amounting to about 50 to 60% of the total cellular DNA in minimal medium and about 120 to 200% in a medium enriched with amino acids. Reshift back to permissive temperature after the inhibition of DNA synthesis at 42° allows for recovery of DNA synthesis after a lag of about 30 min. Protein synthesis is required during that lag for the recovery of DNA synthesis at permissive temperature. The density transfer experiments indicate that in the mutant dna-1 chromosome termini are replicated normally at 42° while the initiation of new rounds of replication is inhibited although the mutation is probably leaky at this temperature. The mutant is hypersensitive to sodium deoxycholate at 42° which suggests alteration of the membrane structure.     

Biogenesis of mitochondria. 53

Summary As shown by gel electrophoresis analysis, E. coli mutant 219 is mutated on the gene coding for S4. This mutant and the parental strain have been studied at the permissive (30°) and the non-permissive temperature (42°) for ribosome assembly and r-protein biosynthesis.The extracts of cells grown at the non-permissive temperature were analyzed by sucrose gradients: Particles sedimenting more slowly (28S) than normal 30S accumulate while 50S precursors undergo maturation and attach to the preformed 30S subunits yielding 70S ribosomes. In addition a small but detectable amount of 30S is also synthesized at 42°. The 28S particles contain all 30S r-proteins except S1, S2 and S12; S5, S7 and S21 are present in reduced amount.The relative rate of biosynthesis of individual r-proteins was determined by pulse-labelling the cells with radioactive leucine. Individual r-proteins were purified from cell extract by the three-dimensional gel electrophoresis technique. The relative rate of biosynthesis of 50S proteins is unchanged in mutant cells grown at 42°. Only the rate of synthesis of five 30S proteins is modified by the temperature shift: S10, S13, S20 and S21 have an increased rate, while S18 is synthesized at a reduced rate. Thus in cells deficient in the assembly of 30S subunits, although the biosynthesis of a few 30S r-proteins is specifically altered, the synthesis of most r-proteins appears to be controlled in the same way as are total cell proteins.     

Intracellular serine proteinase behaves as a heat-stress protein in nongrowing but as a cold-stress protein in growing populations of Bacillus megaterium

A temperature increase from 35° to 40–42°C enhances the rise of cytoplasmic serine proteinase (ISP1) activity in Bacillus megaterium incubated in a sporulation medium. A temperature shift from 27°C in the growth medium to 35°C in the sporulation medium has the same effect. Elevated temperature stimulates the increase of ISP1 level when applied immediately after the transfer of cells from the growth to the sporulation medium (at T₀) or at T₃, when sporulation becomes irreversible. The cytoplasmic PMSF-resistant activity or the proteolytic activity associated with the membrane fraction is stimulated only slightly or not at all. A temperature increase to 45–47°C suppresses the rise of proteolytic activities in all cell fractions. In addition to the elevation of the ISP1 activity by an upward temperature shift, the rise of this enzyme in nongrowing cells is also stimulated by osmotic stress. In growing populations, in contrast to the rise of the ISP1 activity caused by elevated temperature in nongrowing cells, this proteinase is induced by low temperatures (24–27°C). The ISP1 activity roughly correlates with the enzyme protein concentration determined by immunoblotting.     

A cold-adapted extracellular serine proteinase of the yeast<Emphasis Type="Italic"> Leucosporidium antarcticum</Emphasis>

An extracellular serine proteinase, lap2, from the psychrophilic antarctic yeast Leucosporidium antarcticum 171 was purified to homogeneity and characterized. The enzyme is a glycoprotein with a molecular mass of 34.4 kDa and an isoelectric point of pH 5.62. The proteinase is halotolerant, and its activity and stability are dependent neither on Ca²⁺ nor on other metal ions. Lap2 is a true psychrophilic enzyme because of low optimal temperature (25°C), poor thermal stability, relatively small values of free energy, enthalpy and entropy of activation, and high catalytic efficiency at 0–25°C. The 35 N-terminal amino acid residues of lap2 have homology with subtilases of the proteinase K subfamily (clan SB, family S8, subfamily C). The proteinase lap2 is the first psychrophilic subtilase in this family.Communicated by K. Horikoshi     

The proteomic response of sea squirts (genus Ciona) to acute heat stress: a global perspective on the thermal stability of proteins

Congeners belonging to the genus Ciona have disparate distributions limited by temperature. Ciona intestinalis is more widespread with a cosmopolitan distribution ranging from tropical to sub-arctic zones, while Ciona savignyi is limited to temperate-latitudes of the northern Pacific Ocean. To compare the heat stress response between congeners, we quantified changes in protein expression using proteomics. Animals were exposed to 22 °C, 25 °C, and 28 °C for 6 h, then recovered at a control temperature (13 °C) for 16 h (high heat stress experiment). In a second experiment we exposed animals to lower levels of heat stress at 18 °C, 20 °C, and 23 °C, with a 16 °C control. A quantitative analysis, using 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry (with a 69% and 93% identification rate for Ciona intestinalis and Ciona savignyi, respectively), showed changes in a number of protein functional groups, including molecular chaperones, extracellular matrix proteins, calcium-binding proteins, cytoskeletal proteins and proteins involved in energy metabolism. Our results indicate that C. intestinalis maintains higher constitutive levels of molecular chaperones than C. savignyi, suggesting that it is prepared to respond faster to thermal stress. Systematic discrepancies between estimated versus predicted molecular masses of identified proteins differed between protein families and were more pronounced under high heat conditions, suggesting that thermal sensitivities are lower for cytoskeletal proteins and ATP-synthase than for any other protein group represented on 2D gels.     

Alteration of Gene Expression Associated with Abscisic Acid-Induced Chilling Tolerance in Maize Suspension-Cultured Cells

ABA induces chilling tolerance in maize (Zea mays L., cv Black Mexican Sweet) suspension-cultured cells at 28[deg] C when ABA was added to the culture medium at least 6 h prior to chilling (4[deg] C), and this induction can be inhibited by blocking protein synthesis with cycloheximide treatment (Z. Xin, P.H. Li [1992] Plant Physiol 99: 707-711). De novo synthesis of proteins and changes in poly(A+) RNAs were investigated during the ABA induction of chilling tolerance at 28[deg] C as well as during chilling exposure. At 28[deg] C, ABA increased the net synthesis of 11 proteins. Five of these proteins, whose net synthesis was also increased by chilling (4[deg] C), were called group I ABA-induced proteins; the remaining six proteins, whose net synthesis was not altered by chilling, were called group II ABA-induced proteins. Chilling suppressed the net synthesis of three proteins. ABA treatment prior to chilling did not alleviate this suppression. ABA applied at the inception of chilling induced neither chilling tolerance nor accumulation of any of the group II proteins; however, once the group II proteins appeared, they were continually synthesized even in a chilling regimen. ABA induced seven in vitro translation products at 28[deg] C. Three of these products could also be induced by chilling; the remaining four were induced by ABA only at 28[deg] C. These results suggest that ABA-induced alteration of protein synthesis at 28[deg] C is associated with an increased chilling tolerance in maize suspension-cultured cells.     

Relationship between heat-shock protein synthesis and thermotolerance in rainbow trout fibroblasts

Summary The role of heat-shock protein synthesis in the development of thermotolerance by rainbow trout fibroblasts was examined. During the first 6 h after being shifted from 22°C to 28°C, cells of the rainbow trout fibroblast line, RTG-2, rapidly synthesized the major heat-shock proteins (hsps), hsps 87, 70 and 27, and developed tolerance to 32°C. After 24 h at 28°C hsp synthesis was drastically reduced but thermotolerance was maintained. If these thermotolerant cells were shifted to 32°C, hsp synthesis continued at a very low level, but if they were subsequently returned to 22°C, synthesis of hsps 70 and 27 was induced again. The addition of actinomycin D during the first 6 h at 28°C prevented hsp synthesis and the development of thermotolerance. The presence of actinomycin D during the incubation of thermotolerant cultures at 32°C blocked the reinitiation of hsps synthesis at 22°C but had no effect on survival. Therefore, the hsps that accumulated at 28°C were sufficient to allow cells to survive a subsequent thermal stress at 32°C.     

Production of extracellular inulinase in high-cell-density fed-batch cultures of Kluyveromyces marxianus

The production of extracellular inulinase (\-1,2-d-fructan fructanohydrolase, EC 3.2.1.7) was studied in fed-batch cultures of the yeast Kluyveromyces marxianus CBS 6556 at 30 and at 40° C. At both temperatures, the final biomass concentration exceeded 100 g·l^–1 and more than 2 g enzyme. L^–1 of culture supernatant was produced. The biomass yield on O₂ at 40° C was substantially lower than at 30°C. Nevertheless, at 40° C a growth rate of 0.20 h^–1 could be maintained for a longer period than at 30° C. The unexpected higher O₂-transfer rate at 40°C is probably due to a lower viscosity of the culture broth. The 40°C fermentation took only 33 h as compared to 42 h at 30° C. These results indicate that K. marxianus is a promising host for the extracellular production of heterologous proteins under the control of the inulinase promoter.     

Infectivity of Strongyloides venezuelensis is influenced by variations in temperature and time of culture

The present research investigated the influence of temperature and time of larvae culture on the infectivity of Strongyloides venezuelensis. Mice were infected s.c. with 1500 larvae of S. venezuelensis maintained at 28 °C for three days of culture (dc), 28 °C for seven dc or 18 °C for seven dc. On days 1, 3, 5, 7, 14 and 21 post-infection the animals were sacrificed and cell numbers in the blood, peritoneal cavity fluid (PCF), broncoalveolar fluid (BALF), cytokines, immunoglobulins, number of parasites and eggs/g of feces were quantified. Results demonstrated an increase in eosinophils and mononuclear cells in the blood, PCF and BALF of infected mice. Larvae at 28 °C/3dc induced earlier eosinophils in the PCF and BALF as opposed to larvae at 28 °C/7dc and 18 °C/7dc. Larvae at 28 °C/7dc induced higher synthesis of IL-4, IL-5 and IL-10 on days 5 and 7 post-infection. Larvae at 28 °C/3dc in culture induced higher synthesis of IL-12 than larvae of seven dc, but time in culture induced better synthesis of IFN-γ after larval migration had ceased and only adult worms were present. Larvae at 28 °C/3dc in culture induced higher synthesis of IgG and IgG1 and expelled less female parasites than larvae cultivated for seven days. In conclusion, it was observed that the infectivity of S. venezuelensis is influenced by variations in temperature and time of culture.     

Initiation of chromosomal DNA replication which is stimulated without oversupply of DnaA protein in Escherichia coli

Summary The temperature-sensitive dnaA46 mutation in Escherichia coli can be phenotypically suppressed at 42° C by oversupply of GroELS proteins, and the suppressed cells grow extremely slowly at 30° C. We found that the phenotype of dnaA46 showing this cold sensitivity was dominant over the phenotype of dnaA ⁺, and could not be rescued by introduction of oriC-independent replication systems. These results suggest that the cold sensitivity was not caused by a simple defect in replication. When a growing culture of a dnaA46 strain with a GroELS-overproducing plasmid was shifted from 42° to 30° C in the presence of chloramphenicol, the chromosomal DNA replicated excessively. Initiation of replication occurred at the site of oriC repeatedly four or five times during a 4 h incubation period without concomitant protein synthesis, indicating an excessive capacity for initiation. Such overreplication did not take place at 42° C in the suppressed dnaA46 strain, or at either temperature in GroELS-oversupplied dnaA ⁺ cells. No significant difference was detected between the cellular content of DnaA protein in suppressed cells where the initiation capacity was abnormally high, and that in wild-type cells in which the initiation capacity was normal. Thus, DnaA protein might function in vivo through some phase control mechanism for initiation, apart from a simple regulation by its total amount. A possible mechanism is proposed based on the participation of GroELS proteins in protein folding.A preliminary account of this work was presented at the Annual Meeting of the Molecular Biology Society of Japan in 1989.     

Induction of stress proteins inLeuconostoc oenos to perform direct inoculation of wine

Summary The enhancement or induction of the protein synthesis was clearly observed in cells ofL. oenos labeled with³⁵S for five proteins during heat shock at 42°C and acid shock at pH 3. Furthermore, no stress protein was induced after exposure ofL. oenos to ethanol shock 10% (v/v). Moreover, survival ofL. oenos in wine and ability to perform alolactic fermentation was improved after direct inoculation when cells were pretreated at 42°C.     

Extracellular alkaline protease of newly isolatedRhizopus oryzae

Summary A fungal isolate identified asRhizopus oryzae, produces an extracellular alkaline serine protease. Maximum protease formation was after six days in shake flask culture at two different conditions of pH and temperature optimum (pH 5 at 30°C and pH 10 at 37°C). AgNO₃ and Tween 80 increased protease synthesis. The enzyme is stable between pH 3 and pH 11 and has a temperature optimum of 60°C.     

Influence of temperature on the production of an archaeal thermoactive alcohol dehydrogenase from Pyrococcus furiosus with recombinant Escherichia coli

The heterologous production of a thermoactive alcohol dehydrogenase (AdhC) from Pyrococcus furiosus in Escherichia coli was investigated. E. coli was grown in a fed-batch bioreactor in minimal medium to high cell densities (cell dry weight 76 g/l, OD₆₀₀ of 150). Different cultivation strategies were applied to optimize the production of active AdhC, such as lowering the cultivation temperature from 37 to 28°C, heat shock of the culture from 37 to 42°C and from 37 to 45°C, and variation of time of induction (induction at an OD₆₀₀ of 40, 80 and 120). In addition to the production of active intracellular protein, inclusion bodies were always observed. The maximal activity of 30 U/l (corresponding to 6 mg/l active protein) was obtained after a heat shock from 37 to 42°C, and IPTG induction of the adhC expression at an OD₆₀₀ of 120. Although no general rules can be provided, some of the here presented variations may be applicable for the optimization of the heterologous production of proteins in general, and of thermozymes in particular.