Schwanniomyces Castellii Research Articles

Glucose metabolism in the yeast Schwanniomyces castellii: role of phosphorylation site I and an alternative respiratory pathway.

Glucose metabolism in a Crabtree-negative yeast, Schwanniomyces castellii, and a cytochrome b-deficient mutant of this strain was investigated in chemostat culture. The wild-type and mutant strains exhibited the same behavior. Oxidative metabolism was observed when the substrate uptake rate (qS) was low. Fermentative metabolites were excreted when the qS value was higher than 0.40 g.g-1.h-1, indicating the occurrence of a respirofermentative metabolism; however, the respiratory quotient (RQ) remained near 1. When fermentation occurred, the cytochrome pathway was repressed but not the salicylhydroxamic acid (SHAM)-sensitive pathway. The presence of an alternative SHAM-sensitive respiratory pathway and the presence of phosphorylation site I in all metabolic conditions explained the RQ value of 1 and accounted for high biomass yields in oxidative metabolism conditions (0.62 g.g-1 for the wild-type strain and 0.31 g.g-1 for the cytochrome b-deficient mutant strain).

Optimization of single-cell-protein production from cassava starch using Schwanniomyces castellii

Schwanniomyces castellii B5285 grew faster and produced greater biomass and higher protein yield than either S. alluvius ATCC 26074 or S. alluvius 81Y when these amylolytic yeasts were grown with 2% (w/v) cassava starch as sole C source. With 0.5% (w/v) glutamate as N source, S. castellii reached 7.12 g cell dry mass/l, with a protein yield of 6.4 g/100 g starch. The optimal agitation speed, aeration rate and pH for growth of this yeast in a fermenter were 400 rev/min, 1.67 vol./vol.min. and 5.0, respectively. Tween 80 at 0.1% increased cell dry mass to 8.90 g/l, cell yield to 44 g/100 g starch and protein yield to 7.4 g/100 g starch.

Maintenance of heat and water balances as a scale-up criterion for the production of ethanol by schwanniomyces castellii in a solid state fermentation system

The scale-up of column fermenters by 6 to 410 gravimetric scale factors, from 10 g moist substrate size, has been achieved efficiently by maintaining heat and water balances in the media. The data on the patterns of ethanol production, biomass formation, the concentration of different carbohydrates, dry matter and pH values against time were of equal magnitude in 10- and 60-g size column fermenters. The reactors of 60-, 370- and 4100-g sizes also compared well in respect of O 2 consumption, CO 2 evolution and the specific growth rates in aerobic and anaerobic fermentation phases. The overall productivities of ethanol were similar in all the four column fermenters. The ability to obtain the same results in all the fermenter sizes, in spite of the increases in the diameter and the height of the columns, indicates the high potential of this simple scale-up criterion which has not been used earlier for scale-up of any fermentation process.

Alternative respiratory pathways in Schwanniomyces castellii: involvement in energy production and growth.

Wild-type and cytochrome-deficient strains of Schwanniomyces castellii were cultivated aerobically on citrate, and the effects of respiratory inhibitors on respiration, ATP levels and energy charge were measured. The obtained results demonstrate that every respiratory pathway of Schwanniomyces castellii involves phosphorylation site I, and suggest that the functioning of this site allows the alternative pathways to produce ATP.

Transport and hydrolysis of maltose by Schwanniomyces castellii.

Hydrolysis and transport of maltose into Schwanniomyces castellii was studied under aerobic and anaerobic conditions. Amylase and glucosidase were not synthetized in presence of maltose in anaerobic conditions. Maltose permease was synthesized in anaerobiosis and its functioning is not inhibited. Glucose strongly repressed induction. The half-saturation constant for uptake of maltose was 0.06 mM. The rate of uptake of maltose was decreased by 2,4-dinitrophenol, antimycin or sodium azide. The significance of these results in relation to the Kluyver effect is discussed.

Utilization of phytate by some yeasts

Of 21 yeast strains screened for ability to hydrolyse phytic acid salts, nine strains grew on sodium phytate as sole source of inorganic phosphate. Of the five most interesting strains for their growth parameters tested and for their phytase activity in batch-culture,Schwanniomyces castellii CBS 2863 had the highest phytase activity in presence of 5 g phytate I−1.

Expression and regulation of glucoamylase from the yeast Schwanniomyces castellii

Expression of the 146-kilodalton (kDa) extracellular glucoamylase by the budding yeast Schwanniomyces castellii is induced by maltose and starch. By use of antiglucoamylase antisera, we found that this expression was regulated at the level of the mRNA, taking place within 30 min after exposure of yeast cells to the respective sugars. Polyacrylamide gel electrophoresis analysis of the in vitro-translated products of total RNA from maltose-treated cells established that the glucoamylase precursor was approximately 120 kDa in size. Stable glucoamylase transcript was not produced in cells exposed to glucose, 2-deoxyglucose, and heat shock. Cells exposed to these two sugars also degraded intracellular and extracellular glucoamylase. In the presence of sugars such as cellobiose, galactose, lactose, and xylose or in the absence of any carbohydrate, a low-level, constitutive-like expression of this preglucoamylase occurred. The nascent glucoamylase underwent at least two posttranslational modifications, resulting in a 138-kDa cell-associated form and the 146-kDa active form that was found free in the medium. These results suggest that glucoamylase expression is tightly regulated similarly to expression of the enzymes responsible for maltose metabolism in Saccharomyces yeasts.

Alternative respiration pathways in Schwanniomyces castellii

We have isolated and studied cytochromic-deficient mutants of the amylolytic yeast Schwanniomyces castellii in order to study the possible contribution of cytochromes to alternative pathways. Three mutants were found, lacking cytochrome b, a + a3, or b and a + a3. All strains presented two alternative pathways, which were induced in the wild strain when cytochromic respiration was suppressed by growth in the presence of inhibitors, or without copper. If cytochromic respiration was absent, the Yxs yields in aerobiosis were higher than in anaerobiosis. This shows that the alternative pathways play a part in energy conservation. Cytochrome a + a3 did not appear to be directly involved in the alternative pathways.

Isolation and characterization of a hexokinase mutant of Schwanniomyces castellii: Consequences on cell production in continuous culture

AbstractA mutant of Schwanniomyces castellii with reduced glucose phosphorylation and with practically no phosphorylation of fructose was investigated. Carbon catabolite represion of α‐glucosidase and amylases was reduced. Repression of β‐galactosidase was normal. We have compared in continuous culture this mutant strain with wild type and another previously described mutant. The relationship between the specific rate of glucose consumption (Qs) and residual glucose concentration (s) in an inverse mode, suggests that there may be two types of transport of glucose. Mutation at the phosphorylation level causes apparent modification of the kinetic parameters of glucose uptake rate. The consequence of mutation at the phosphorylation level on biomass production was discussed.

Isolation and characterization of a derepressed mutant of Schwanniomyces castellii for amylase production

The production of α-amylase and amyloglucosidase activity in the yeast Schwanniomyces castellii strain CBS 2863 is repressed in the presence of glucose. Mutants displaying increased amylase activity were obtained after treatment with UV light and screening for resistance to 2-deoxy-glucose. One mutant was found to exhibit derepressed amylase activity. Biosynthesis and the rate of excretion did not appear to be as highly sensitive to dissolved oxygen, pH and dilution rate as in the parental strain.

Transformation of Schwanniomyces occidentalis with an ADE2 gene cloned from S. occidentalis.

We have developed an efficient transformation system for the industrial yeast Schwanniomyces occidentalis (formerly Schwanniomyces castellii). The transformation system is based on ade2 mutants of S. occidentalis deficient for phosphoribosylaminoimidazole carboxylase that were generated by mutagenesis. As a selectable marker, we isolated and characterized the S. occidentalis ADE2 gene by complementation in an ade2 strain of Saccharomyces cerevisiae. S. occidentalis was transformed with the recombinant plasmid pADE, consisting of a 4.5-kilobase-pair (kbp) DNA fragment from S. occidentalis containing the ADE2 gene inserted into the S. cerevisiae expression vector pYcDE8 by a modification of the spheroplasting procedure of Beggs (J. D. Beggs, Nature [London] 275:104-108, 1978). Intact plasmids were recovered in Escherichia coli from whole-cell lysates of ADE+ transformants, indicating that plasmids were replicating autonomously. High-molecular-mass species of pADE2 were found by Southern hybridization analysis of intact genomic DNA preparations. The shift to higher molecular mass of these plasmids during electrophoresis in the presence ethidium bromide after exposure to shortwave UV suggests that they exist in a supercoiled form in the transformed host. Subclones of the 4.5-kbp insert indicated that ADE2-complementing activity and sequences conferring autonomous replication in S. occidentalis were located within a 2.7-kbp EcoRI-SphI fragment. Plasmids containing this region cloned into the bacterial vector pUC19 complemented ade2 mutants of S. occidentalis with efficiencies identical to those of the original plasmid pADE.

Differential glucoamylase expression in Schwanniomyces castellii induced by maltose

Different levels of glucoamylase expression occurred when Schwanniomyces castellii strain 1402 was shifted during growth on glucose to either glucose, maltose, or soluble starch medium. Extracellular glucoamylase activity was greatest from cells grown on maltose (~22×), slightly less on soluble starch (~16×), and least on glucose (1×). Glucoamylase biosynthesis was further studied by labelling of total proteins in vivo with [35S]methionine and immunoprobing with a polyclonal anti-glucoamylase antibody. Mature active glucoamylase is 146 kDa. Maltose cultures expressed four cellular (75, 78,138, and 146 kDa) and two extracellular (78 and 146 kDa) polypeptides. Neither the 138- nor the 146-kDa products were detected in cells in the presence of glucose; the 78-kDa product is expressed at approximately 5% the level obtained from cells in maltose. The 146-kDa glucoamylase is expressed within 30 min after transfer of cells from glucose- to maltose-containing medium. This expression appears to be cell growth and concentration independent and is therefore similar to galactose-inducible enzyme expression in Saccharomyces cerevisiae.

Influence of culture conditions on the cell yield and amylases biosynthesis in continuous culture by Schwanniomyces castellii

Schwanniomyces castellii excreted α-amylase and amyloglucosidase into the medium in the presence of starch. The biosynthesis and the rate of excretion were influenced by dissolved oxygen (specially for α-amylase), pH of the culture and dilution rate. The cell yield observed (0.59) remained constant up to D=0.35h-1 with starch as substrate. But in the case of growth on glucose, the yield observed was equal to 0.62 up to a dilution rate of D=0.18 h-1. Beyond this value Y x/s decreased and ethanol was produced. The onset of fermentation dependend partly on the nature of the substrate and not only on the environment in particular on the quantity of dissolved oxygen present.

Genetic analysis of intergeneric hybrids obtained by protoplast fusion in yeasts

Prototrophic hybrids have been obtained by the fusion of various auxotrophic haploid strains of Saccharomyces cerevisiae and Schwanniomyces castellii. The fusion hybrids showed starch fermentability which derived from one of the fusion parents, S. castellii. Surprisingly, these fusion hybrids were found to exhibit excellent sporulation and spore germination. The progenies of these fusion hybrids showed a few aberrant segregations, but mostly normal segregation for auxotrophic genetic markers. They also showed many tetrads with an apparently digenic segregation (2:2, 3:1 and 4:0) for starch fermentation. On the other hand, mating types of segregants of the fusion hybrids were determined by the prototrophic recovery method. Consequently, tetrad types for mating type were mostly 2a:1 alpha: 1 non-mater and several asci showed tetrad types of 2a: 2 non-mater and 2a:2 alpha. The 60 prototrophic fusion hybrids and its segregants did not secrete alpha-amylase on the starch agar plate. However, all of the data suggested that fusion hybrid could carry two dominant genes (STAB and STAC) to ferment starch, and that the two genes STAB and STA2 may be identical or allelic as may be the genes STAC and STA3.

Ethanol inhibition of growth, fermentation and starch hydrolysis in Schwanniomyces castellii

The inhibitory effect of ethanol on several parameters of starch fermentation was studied by comparing the strain Schwanniomyces castellii and the corresponding respiratory-deficient mutant. The effect of ethanol on cell viability and cell growth depends on the yeast metabolism. Inhibition was greater when the fermentative metabolism was preponderant. Amylases were shown to be inhibited at ethanol concentrations which are often obtained in fermentation. This inhibition conformed to non-competitive kinetics and was reversible. Enzyme biosynthesis was reduced in the presence of ethanol.

Direct alcoholic fermentation of starchy biomass using amylolytic yeast strains in batch and immobilized cell systems

Direct alcoholic fermentation of dextrin or soluble starch with selected amylolytic yeasts was studied in both batch and immobilized cell systems. In batch fermentations, Saccharomyces diastaticus was capable of fermenting high dextrin concentrations much more efficiently than Schwanniomyces castellii. From 200 g·l−1 of dextrin S. diastaticus produced 77 g·l−1 of ethanol (75% conversion efficiency). The conversion efficiency decreased to 59% but a higher final ethanol concentration of 120 g·l−1 was obtained with a medium containing 400 g·l−1 of dextrin. With a mixed culture of S. diastaticus and Schw. castellii 136 g·l−1 of ethanol was produced from 400 g·l−1 of dextrin (67% conversion efficiency). S. diastaticus cells attached well to polyurethane foam cubes and a S. diastaticus immobilized cell reactor produced 69 g·l−1 of ethanol from 200 g·l−1 of dextrin, corresponding to an ethanol productivity of 7.6g·l−1·h−1. The effluent from a two-stage immobilized cell reactor with S. diastaticus and Endomycopsis fibuligera contained 70 g·l−1 and 80 g·l−1 of ethanol using initial dextrin concentrations of 200 and 250 g·l−1 respectively. The corresponding values for ethanol productivity were 12.7 and 9.6 g·l−1·h−1. The productivity of the immobilized cell systems was higher than for the batch systems, but much lower than for glucose fermentation.

New Developments in Ethanol Fermentation

Production of ethanol by microorganisms, as a result of the fermentation of substrates such as sugars or starch, is a process that predates recorded history. Although a number of microorganisms are able to produce ethanol, more than 96% of the fermentation ethanol that is produced today employs the yeast Saccharomyces cerevisiae or its related species. Such yeast strains must be able to ferment a wide range of sugars, and yeast strains possessing amylolytic ability are becoming important as producers of fermentation ethanol from starch/dextrin-containing substrates such as wort. A brewing yeast strain must effectively remove the desired nutrients from the growth medium (i.e., the wort), must impart the required flavor to the beer, and must be effectively removed from the fermented wort after the cultures have fulfilled their metabolic role. In the normal situation, brewer's yeast strains are incapable of fermenting dextrin material; however, yeast strains are now available that are capable of fermenting at least a part of this dextrin material and producing a palatable beer. A process is described that produces a low carbohydrate beer employing amylolytic enzymes of a derepressed Schwanniomyces castellii strain able to overproduce α-amylase and glucoamylase with debranching ability.

Characterization of Schwanniomyces castellii mutants with increased productivity of amylases

The production of α-amylase activity in the yeast Schwanniomyces castellii strain 1402 is repressed in the presence of the non-metabolizable glucose analogue, 2-deoxy-glucose. Selection for resistance to 2-deoxy-glucose after treatment with ethyl methane sulphonate (EMS) or UV light has yielded mutants displaing increased α-amylase activities. One such mutant, S. castellii strain 1436, was found to exhibit constitutive α-amylase activity in glucose-containing medium. This constitutive enzyme activity was also observed under pilot scale fermentation conditions when the pH was maintained constant at 5.5±0.1. The disaccharide maltose served as a stronger inducer of α-amylase activity than the natural substrate starch in both the wild type (1402) and mutant (1436) strains.

Susceptibility of Saccharomyces spp. and Schwanniomyces spp. to the aminoglycoside antibiotic G418.

Industrially useful polyploid yeasts such as the brewing yeasts do not possess any auxotrophic genetic markers and hence are not easily amenable to plasmid-mediated DNA transformations. In an attempt to obtain genetic markers, a number of useful Saccharomyces sp. strains and some amylolytic Schwanniomyces sp. strains were tested for their susceptibility to the antibiotic Geneticin G418 , a 2-deoxystreptamine reported to be active against bacteria, yeasts, and plant and animal cells. All of the Saccharomyces sp. strains, including the brewing strains, were found to be susceptible to G418 in the concentration range of 150 to 500 micrograms/ml. Of the three Schwanniomyces species investigated, only Schwanniomyces castellii (strain 1402) was found to be resistant to G418 at concentrations up to 1 mg/ml. Resistance was exhibited both in liquid media and on glycerol-peptone-yeast extract agar plates. This finding is interesting in view of the possibility of using this strain as a DNA donor for transformations aimed at introducing the amylolytic capability into brewing yeasts.

Effects of pretreatments with sulfhydryls and alkylating agents on spheroplast formation from Schwanniomyces species

Pretreatment of cells with β-mercaptoethanol, dithiothreitol, or cysteine increased the rate of spheroplast formation at all stages of growth in Schwanniomyces castellii and S. occidentalis, but had little effect on final yields of spheroplasts when compared with controls. Pretreatment with iodoacetate and cystine resulted in decreased rates of formation and lower yields of spheroplasts at all stages of growth for both species. The enhanced rates of spheroplast formation in the presence of the sulfhydryl compounds were more pronounced when the experimental cells were derived from early or late stationary phase cultures, whereas the inhibitory effects of cystine and iodoacetate were not associated with the growth phase. In agreement with extant data on other yeast genera, sulfhydryl compounds increased the susceptibility of the yeast cell wall to degradative enzymes, whilst alkylating agents decreased susceptibility of cell walls to these enzymes.