Construction of a Stable α-Galactosidase-Producing Baker's Yeast Strain

Molasses is widely used as a substrate for commercial yeast production. The complete hydrolysis of raffinose, which is present in beet molasses, by Saccharomyces strains requires the secretion of α-galactosidase, in addition to the secretion of invertase. Raffinose is not completely utilized by commercially available yeast strains used for baking, which are Mel⁻. In this study we integrated the yeast MEL1 gene, which codes for α-galactosidase, into a commercial mel⁰ baker's yeast strain. The Mel⁺ phenotype of the new strain was stable. The MEL1 gene was expressed when the new Mel⁺ baker's yeast was grown in molasses medium under conditions similar to those used for baker's yeast production at commercial factories. The α-galactosidase produced by this novel baker's yeast strain hydrolyzed all the melibiose that normally accumulates in the growth medium. As a consequence, additional carbohydrate was available to the yeasts for growth. The new strain also produced considerably more α-galactosidase than did a wild-type Mel⁺ strain and may prove useful for commercial production of α-galactosidase.     

Fructose metabolism in Zymomonas mobilis

Summary In the metabolism of fructose by Zymomonas, the ethanol yield is decreased due to the formation of dihydroxyacetone, mannitol and glycerol. The reduction of fructose to mannitol by an NADPH-dependent mannitol dehydrogenase is apparently coupled to the oxidation of glucose-6-phosphate by glucose-6-phosphate dehydrogenase, which exhibits higher activity with NADP than with NAD as cofactor. The relatively low cell yield on fructose can partly be explained by the loss of ATP in the formation of dihydroxyacetone and glycerol and partly by the toxic effect of dihydroxyacetone and acetaldehyde on the growth of the organism.     

Production of cyclomaltodextrin glucanotransferase of Bacillus circulans var. alkalophilus ATCC21783 in B. subtilis

Summary The cyclomaltodextrin glucanotransferase (CGTase, E.C. 2.4.1.19) gene from an alkalophilic Bacillus circulans var. alkalophilus ATCC21783 was cloned into Escherichia coli and B. subtilis. When cloned from E. coli to B. subtilis, the entire insert containing the CGTase gene was, depending on the plasmid construction, either unstable or the recombinant B. subtilis did not secrete the enzyme in significant amounts. To achieve efficient enzyme production in B. subtilis, the gene was placed under the control of the B. amyloliquefaciens -amylase promoter. In one of the constructions, both the promoter and the signal sequence of the gene were replaced with those of B. amyloliquefaciens, whereas in another construction only the promoter area was exchanged. The recombinant B. subtilis clones transformed with these plasmid constructions secreted CGTase into the culture medium 14 times as much as did the parental strain in shake flask cultures. In fermentor cultures in an industrially feasible medium the enzyme production was substantially higher, yielding 1.2 g/l of CGTase, which is about 33 times the amount of the enzyme produced by the parental strain in corresponding fermentations. Both of the plasmid constructions were stable when grown over 50 generations without antibiotic selection.     

A new family of polymorphic metallothionein-encoding genes MTH1 (CUP1) and MTH2 in Saccharomyces cerevisiae.

By pulsed-field gel electrophoresis of chromosomal DNA and hybridization with a cloned MTH1 (CUP1) gene, we determined the locations of metallothionein-encoding gene sequences on chromosomes in monosporic cultures of 76 natural strains of Saccharomyces cerevisiae. Most of the strains (68) exhibited a previously known location for the MTH sequence on chromosome (chr.) VIII. Seven strains (resistant or sensitive to Cu2+) showed a MTH sequence in a new locus, MTH2, on chr. XVI. One strain carried an MTH locus on both chromosomes VIII and XVI. Restriction fragment and Southern blot analyses showed that the two MTH loci were very closely related. The strains displayed heterogeneity in the size and structure of their MTH2 locus. The length of the repeat unit of MTH2 varied: a 1.9-kb or 1.7-kb unit was found, instead of the 2.0-kb unit of the MTH1 locus. The most resistant strain (resistant to 1.2 mM CuSO4) contained a 0.9-kb repeat unit in addition to those of 1.9 kb and 1.7 kb. All three sensitive (to over 0.3 mM CuSO4) strains with an mth2 locus had a repeat unit of 1.9 kb or 1.7 kb, suggesting the presence of at least two copies of the MTH2 gene, with one always being in the junction area outside of the repeat unit. A monogenic tetrad segregation of 2:2 was usually found in crosses of resistant MTH2 and sensitive mth2 strains. Hybrids between strains with different MTH loci in all combinations showed low ascospore viability, suggesting that the complete lack of an MTH locus may lead to the death of segregants on YPD medium. The MTH1 and MTH2 loci were exchangeable. Strains with a high level of Cu2+ resistance were also resistant to Cd2+. However, these two properties did not cosegregate in heterozygotic hybrids.     

Partial characterization of a new C3-type capsule-dissolving phage of Streptococcus cremoris.

A viscous, ropy, sour milk product, called 'viili,' is produced in Finland. Capsule-forming strains of Streptococcus cremoris are the typical starters for this product. Occasionally fermentation fails and results in a non-ropy clot. The reasons for these failures, however, are obscure. In one batch of spoiled 'viili,' a new C3-type bacteriophage, termed KSY1, was isolated. The head of the phage was about 230 nm long and about 50 nm wide and the tail was 35 nm long and carried a complex collar structure. Upon infection of a number of encapsulated cultures of S. cremoris with KSY1, the cocci, though not serving as a host of the phage, lost their capsules. A capsuleless strain, S. cremoris 249, served as a host. The latent period was about 150 min and the average burst size 80. The bouyant density of KSYI1 was 1.436 g/cm3.     

Physical and chemical characteristics of shallow embayments on the southern coast of Finland

Regular water-quality monitoring is yielding a great number of hydrographical and chemical data regarding the brackish Baltic Sea. However, present monitoring programmes often do not include shallow and sheltered near-shore areas, which are adjacent to human population and used for recreation purposes. We collected new near-shore water quality data from the southern coast of Finland that will allow the definition of the present state of these systems and may serve as reference data for possible future monitoring programmes. The data set comprises 45 small and shallow embayments with a mean surface area of 211 ha and a mean depth of 4.3 m. According to redundancy analysis (RDA), catchment land use explained 20.3% of the variance in the measured water chemistry. Agriculture had the strongest influence on the water quality of the study sites, capturing 13.1% of the variance. Nitrogen seemed to be strongly related to agriculture, whereas phosphorus was correlated with urban areas and industry. Higher than average nutrient concentrations regularly occurred at the mouths of large rivers, or close to human settlement, emphasising the significance of local sources to the nutrient concentrations of coastal waters. No increasing trend in nutrient or chlorophyll-a concentrations was observed from west to east in these shallow and sheltered nearshore areas. This contrasts with earlier results from pelagic samplings that report a marked influence of the River Neva and St. Petersburg on the nutrient status of the Gulf of Finland. Because these shallow embayments are strongly influenced by the local nutrient load, continued measures must be undertaken to reduce the nutrient load from land to prevent the continued deterioration and eutrophication of the nearshore, coastal waters.     

Marked early 20th century pollution and the subsequent recovery of Töölö Bay, central Helsinki, as indicated by subfossil diatom assemblage changes

Diatoms were used to infer the trophic status history of a Baltic Sea inlet, Töölö Bay, located in central Helsinki. The waterbody received partially treated sewage effluent from the local sewage works and from a sugar factory from ca. 1890 until ca. 1960 when primary sewage treatment and the diversion of the effluent began. The increased nutrient input is shown in the diatom stratigraphy in the form of a rapid increase in planktonic eutrophic forms particularly Cyclotella cf. meneghiniana, and a reciprocal destruction of the periphytic flora. The overall diversity decreased markedly at this time. There is a gradual recovery of the system to the predisturbance conditions from 1960 onwards, as indicated by the re-emergence of the mesotrophic periphytic diatoms. Detrended correspondence analysis (DCA) is used to summarize the responses of the diatom assemblages to changing nutrient concentrations.     

Construction of a Stable alpha-Galactosidase-Producing Baker's Yeast Strain

Molasses is widely used as a substrate for commercial yeast production. The complete hydrolysis of raffinose, which is present in beet molasses, by Saccharomyces strains requires the secretion of alpha-galactosidase, in addition to the secretion of invertase. Raffinose is not completely utilized by commercially available yeast strains used for baking, which are Mel. In this study we integrated the yeast MEL1 gene, which codes for alpha-galactosidase, into a commercial mel baker's yeast strain. The Mel phenotype of the new strain was stable. The MEL1 gene was expressed when the new Mel baker's yeast was grown in molasses medium under conditions similar to those used for baker's yeast production at commercial factories. The alpha-galactosidase produced by this novel baker's yeast strain hydrolyzed all the melibiose that normally accumulates in the growth medium. As a consequence, additional carbohydrate was available to the yeasts for growth. The new strain also produced considerably more alpha-galactosidase than did a wild-type Mel strain and may prove useful for commercial production of alpha-galactosidase.     

The distribution and diversity of Chironomidae (Insecta: Diptera) in western Finnish Lapland, with special emphasis on shallow lakes

Aim Predictions of aquatic ecosystem change with global warming require basic data that accurately reflect the environmental conditions underlying species distributions. However, in remote arctic areas such baseline data are scarce. We assess the influence of environmental variables on chironomid distribution and taxon richness in shallow, isothermal lakes in a poorly studied arctic region. We pay particular attention to community variation along the treeline ecotonal zone where many environmental variables change abruptly in a relatively small area. Location Lake transect in Finnish Lapland spanning from boreal coniferous forest to arctic tundra. Methods Chironomid assemblages were determined from surface‐sediment samples of 50 shallow (< 10 m) natural lakes. Abundance and taxon richness data were related to 24 limnological variables using canonical ordination techniques (DCA, CCA, RDA). A Monte Carlo permutation procedure was used to assess the explanatory power of single variables. Between‐vegetation zone differences of richness were tested for statistical significance using one‐way anova . Results In total, 7771 chironomid head capsules were identified, consisting of 13 species, 10 species groups, four subgenera, 41 genera, four genus groups, five types and three with uncertain taxonomic affiliation. A hump‐shaped relationship between taxon richness and elevation was noted along the study transect with a peak in taxon richness occurring in mountain birch woodland lakes at middle elevations, decreasing then towards both warmer and colder ends of the elevation/temperature gradient. Of the individual parameters, sediment organic content, total organic carbon, pH, and lake‐specific air temperature accounted for the greatest amount of variation in the chironomid data. Main conclusions Maximum taxon richness occurred at mid‐elevations where aquatic algae also reached their maximum diversity. This area coincides with an ecotonal transitional zone, which seems more likely to account for the peak in species richness. Our study demonstrates that the factors most strongly affecting chironomids in Finnish Lapland (i.e. temperature, and ecosystem features) are those that with great probability will also change as a result of future climate change. This will likely have an effect on the distribution of chironomids in subarctic and arctic areas.