Spheroplast fusion of a brewing yeast strain with Saccharomyces diastaticus

Summary One haploid and one diploid strain of Saccharomyces diastaticus carrying genes responsible for glucoamylase synthesis were fused with a brewing polyploid Saccharomyces uvarum lager strain. With the spheroplast fusion technique, the ability to use dextrin and starch was introduced in the brewing yeast. Spheroplasts of the strains to be used were obtained by enzymatic digestion of the cell walls. Fusion took place in polyethylene glycol; complete cells were then regenerated in hypertonic medium containing 3% agar at 37°C. In the first fusion experiment melibiose was used as carbon source; in the second fusion experiment glycerol was employed as carbon source, for the parental Saccharomyces diastaticus diploid strain was a petite mutant. Fusion products were capable of utilizing melibiose and dextrin as carbon sources.     

Use of the somatic fusion method to introduce the flocculation property into Saccharomyces diastaticus

Summary Spheroplasts of a petite mutant of the amylolitic Saccharomyces diastaticus 1376 yeast strain were successfully fused with spheroplasts of a flocculent and respiratory competent Saccharomyces cerevisiae 1161 yeast strain.Flocculent and non-flocculent stable recombinants were recovered after regeneration of the cell walls all of which formed halos around their colonies in media containing starch/dextrin as carbon source. The sporulation ability varied in some of the fusion products and the possible influence of genetic instability is discussed.     

Efficiency of genetically engineered yeast in the production of ethanol from dextrinized cassava starch

Summary The ability of a polyploid/aneuploidSaccharomyces diastaticus spheroplast fusion product and a diploidSaccharomyces diastaticus hybridization product, to produce ethanol from dextrinized cassava starch with varying amounts of supplemented glucoamylase (amyloglucosidase), was investigated. It was found that the added glucoamylase could be reduced by over 50% using these glucoamylase producing strains as compared to a commercially availableSaccharomyces cerevisiae strain commonly used in ethanol producing industries.     

Alcoholic fermentation of starch containing media using yeast protoplast fusion products

Summary Fermentation of starch based industrial media was tested with yeast fusion products previously described, from a Baker's yeastSaccharomyces cerevisiae and Saccharomyces diastaticus and from a highly flocculentSaccharomyces cerevisiae andSaccharomyces diastaticus. The (somatic) fusion products were capable to produce more ethanol than parental strains after 96 h of batch fermentation. The aim of this work was to reduce the amount of enzyme used in saccharification by using good fermenting amylolytic yeast strains.     

Allelism within the DEX and STA gene families in Saccharomyces diastaticus

Summary Saccharomyces diastaticus produces an extracellular glucoamylase and is therefore capable of hydrolyzing and fermenting starch. Tamaki (1978) studied starch utilization in S. diastaticus and found three polymeric genes controlling this function: STA1, STA2 and STA3. Independently, Erratt and Stewart (1978) studied dextrin utilization by the yeast S. diastaticus and designated the gene, which they identified, DEX1. Erratt and Stewart (1981a, b) later described two other genes which controlled glucoamylase production in S. diastaticus: DEX2 and a third which was allelic to STA3. At that time STA1 and STA2 were not available to test for allelism in the DEX gene family. In this study strains containing the remaining 4 genes have been examined to determine if further allelism exists between the two gene families. It was ascertained that DEX1 is allelic to STA2 and DEX2 is allelic to STA1. Therefore, no new gene controlling starch utilization has been identified and these two nomenclatures can now be consolidated into one. Based on the fact that the glucoamylase from S. diastaticus can hydrolyze both dextrin and starch, dextrin being the term used to described partially hydrolyzed starch, and the more wide use of the nomenclature STA, we propose to retain STA as the designation for genes coding for glucoamylase production in S. diastaticus.     

Genetic properties of abortive products resulting from the protoplast fusion in yeasts

Summary Protoplast fusion was carried out by using a routine technique with various auxotrophic strains of Saccharomyces diastaticus and Schizosaccharomyces pombe, and abortive fusion products were observed as small colonies which appeared more frequently than large prototrophic colonies. Sixty abortive fusion products retained one or more auxotrophic characters derived from S. diastaticus, one of the strains used in the protoplast fusion. Several hybrids were obtained between the abortive products and S. cerevisiae, and the segregants of these hybrids showed many aberrant tetrads with regard to some genetic markers. These segregation patterns would be likely to result if the segregating characters were in the trisomic condition +/+/-. The results indicate that (1) the abortive fusion products are an alien monosome additional (AMA) haploid containing the genome of S. diastaticus and only one chromosome of S. pombe. (2) The additional chromosome of S. pombe, which is integrated with the genome of Saccharomyces, can be stably transmitted to the progeny.     

Development of Rapidly Fermenting Strains of Saccharomyces diastaticus for Direct Conversion of Starch and Dextrins to Ethanol

Alcoholic fermentation, growth, and glucoamylase production by 12 strains of Saccharomyces diastaticus were compared by using starch and dextrins as substrates. Haploid progeny produced from a rapidly fermenting strain, SD2, were used for hybridization with other S. diastaticus and Saccharomyces cerevisiae haploids. Alcoholic fermentation and enzyme production by hybrid diploids and their haploid parents were evaluated. Although the dosage of the STA or DEX (starch or dextrin fermentation) genes may enhance ethanol production, epistatic effects in certain strain combinations caused decreases in starch-fermenting activity. Both the nature of the starch or dextrin used and the fermentation medium pH had substantial effects on alcohol production. Commercial dextrin was not as good a substrate as dextrins prepared by digesting starch with α-amylase. Crude manioc starch digested by α-amylase was fermented directly by selected hybrids with almost 100% conversion efficiency. The manioc preparation contained adequate minerals and growth factors. This procedure should be suitable for direct commercial application in manioc-producing regions in Brazil and elsewhere. A rapidly fermenting haploid strain, SD2-A8, descended from strain SD2, contains two unlinked genes controlling formation of extracellular amylase. A convenient method for detecting these genes (STA genes) in replica plates containing large numbers of meiotic progeny was developed.     

Increased osmotolerance of genetically modified ethanol producing strains of Saccharomyces Sp.

Summary A stable spheroplast fusion product of the polyploid brewing strain Saccharomyces uvarum (cares bergensis) , strain 21 and a genetically constructed diploid Saccharomyces diastaticus, strain 1384 has been shown to have improved ethanol producing capability in defined media (Panchal et al., 1982). This fusion product, strain 1400 was further subjected to fermentations in defined media containing glucose substrate and varying concentrations of the non-metabolized sugars sorbitol or mannitol.While the fermentation efficiencies of all the three strains decreased with increasing osmotic pressure imparted by sorbitol or mannitol, the detrimental effect was least apparent with the fusion product than with either of the fusion partners. This attribute of the stable fusion product has major significance in relation to its potential for industrial ethanol production.     

Expression and secretion of human lipocortin-1 by promoter and signal sequence of STA1 from Saccharomyces diastaticus

Summary For the secretion of human lipocortin-1 (LC-1) in yeast, a expression and secretion vector was constructed by using the promoter and signal sequence of glucoamylase gene (STA1) of Saccharomyces diastaticus. After the cDNA of human LC-1 was ligated with the secretion vector, the resulting hybrid plasmid was transformed into S. diastaticus. When the recombinant S. diastaticus was cultivated in YPD medium, LC-1 was expressed and secreted into the extracellular medium, yielding LC-1 protein at a concentration of 2.5 g/mL.     

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

Summary 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.     

Protoplast fusion between a petite strain ofCandida utilis andSaccharomyces cerevisiae respiratory-competent cells

Prototrophic RD mutant cells ofCandida utilis NRRL-Y-1084 and auxotrophic mutant respiratory-competent cells ofSaccharomyces cerevisiae 4003-5Ba his ₄ leu ₂ can ^S meth ₂ trp ₅ ade ₁ ura ₃ gal were turned into protoplasts to be further fused with the aid of polyethylene glycol (PEG) and Ca²⁺ ions. Minimal medium containing glycerol as the carbon source was employed for fusion product selection. The respiratory-competent fusion products, mainly oval cells, resembledCandida utilis and had the fermentative abilities of this strain (dextrose, sucrose, raffinose). Five fusion products were analyzed as to their ability to metabolize dextrose, xylose, cellobiose, trehalose, glycerol, succinic acid, citric acid, salycin, and maltose. Fusion products partially restored the respiratory-competentCandida utilis capacity to grow by use of these carbon compounds, and none of theSaccharomyces cerevisiae fermenting abilities were found. Our results would suggest either a partial recombination between parental mitochondria or some occurring phenomenon affecting the cell, membrane function after somatic fusion without concomitant nuclear fusion.     

Xylulose fermentation by Saccharomyces cerevisiae and xylose-fermenting yeast strains

Xylulose fermentation by four strains of Saccharomyces cerevisiae and two strains of xylose-fermenting yeasts, Pichia stipitis CBS 6054 and Candida shehatae NJ 23, was compared using a mineral medium at a cell concentration of 10 g (dry weight)/l. When xylulose was the sole carbon source and fermentation was anaerobic, S. cerevisiae ATCC 24860 and CBS 8066 showed a substrate consumption rate of 0.035 g g cells^–1 h^–1 compared with 0.833 g g cells^–1 h^–1 for glucose. Bakers' yeast and S. cerevisiae isolate 3 consumed xylulose at a much lower rate although they fermented glucose as rapidly as the ATCC and the CBS strains. While P. stipitis CBS 6054 consumed both xylulose and glucose very slowly under anaerobic conditions, C. shehatae NJ 23 fermented xylulose at a rate of 0.345 g g cells^–1 h^–1, compared with 0.575 g g cells^–1 h^–1 for glucose. For all six strains, the addition of glucose to the xylulose medium did not enhance the consumption of xylulose, but increased the cell biomass concentrations. When fermentation was performed under oxygen-limited conditions, less xylulose was consumed by S. cerevisiae ATCC 24860 and C. shehatae NJ 23, and 50%–65% of the assimilated carbon could not be accounted for in the products determined.     

Expression of Bacillus macerans cyclodextrin glucanotransferase gene in Saccharomyces cerevisiae

Cyclodextrin glucanotransferase (CGTase) gene of Bacillus macerans was subcloned down-stream of yeast ADH1 promoter and expressed in Saccharomyces cerevisiae. Most of the CGTase expressed was in the extracellular medium with a maximum activity of about 0.28 unit ml^–1 after 48 h cultivation. The recombinant CGTase was secreted as an N-linked-glycosylated form and predominantly produced -cyclodextrin from starch.     

Engineering of Cyclodextrin Glucanotransferase on the Cell Surface of Saccharomyces cerevisiae for Improved Cyclodextrin Production

The cyclodextrin glucanotransferase (CGTase) gene (cgt) from Bacillus circulans 251 was cloned into plasmid pYD1, which allowed regulated expression, secretion, and detection. The expression of CGTase with a-agglutinin at the N-terminal end on the extracellular surface of Saccharomyces cerevisiae was confirmed by immunofluorescence microscopy. This surface-anchored CGTase gave the yeast the ability to directly utilize starch as a sole carbon source and the ability to produce the anticipated products, cyclodextrins, as well as glucose and maltose. The resulting glucose and maltose, which are efficient acceptors in the CGTase coupling reaction, could be consumed by yeast fermentation and thus facilitated cyclodextrin production. On the other hand, ethanol produced by the yeast may form a complex with cyclodextrin and shift the equilibrium in favor of cyclodextrin production. The yeast with immobilized CGTase produced 24.07 mg/ml cyclodextrins when it was incubated in yeast medium supplemented with 4% starch.     

Mating Signals Control Expression of both Starch Fermentation Genes and a Novel Flocculation Gene FLO 8 in the Yeast Saccharomyces

In the yeast Saccharomyces diastaticus, expression of both glucoamylase-producing (STA) genes and a novel flocculation gene FLO 8 was greatly diminished by the mating-type locus MATa/MAT α.     

Ethanol production and fermentation characteristics of recombinant saccharomyces cerevisiae strains grown on starch

The production of ethanol from starch has been investigated in three genetically modified Saccharomyces cerevisiae strains (YPG/AB, YPG/MM, and YPB-G). Two of the three strains produce the Aspergillus awamori glucoamylase together with either the Bacillus subtilis (YPG/AB) or the mouse (YPG/MM) α-amylase as separately secreted polypeptides. YPB-G, on the other hand, secretes a bifunctional fusion protein that contains both the B. subtilis α-amylase and the A. awamori glucoamylase activities. Substrate utilization, biomass growth, and ethanol production were all studied in both starch- and glucose-containing media. Much higher growth rates were found when any of the three strains were grown on glucose. YPG/AB showed the most efficient utilization of starch for ethanol production with the lowest levels of reducing sugars accumulating in the medium. The superior performance of YPG/AB as compared to YPB-G was found to correlate with its higher level of α-amylase activity. The ethanol production levels of YPG/AB in starch- and glucose-containing media were found to be comparable. YPB-G, which secretes the bifunctional fusion protein, could produce ethanol in media with starch concentrations above 100 g l⁻¹ while YPG/MM did not produce ethanol from starch because of its negligible secretion of glucoamylase.     

Accelerated ethanol fermentation by Saccharomyces cerevisiae with addition of activated carbon

Fermentation with the addition of activated carbon at 100 g l^–1 promoted the glucose consumption and ethanol production rates of Saccharomyces cerevisiae by 1.3 and 1.1 times, respectively. With fermentation using spent medium, the consumption rate was maintained at 90% of that in the fresh medium with the addition of activated carbon, while the rate without any addition decreased to about 70%.     

Analysis of hybrids obtained by rare-mating of Saccharomyces strains

Summary Rare-mating of closely related Saccharomyces cerevisiae and S. diastaticus strains led to the formation of different hybrids. Mating-type switching and chromosome losses could be observed by means of classical genetic analysis and pulsed field gel electrophoresis of intact chromosomes. The latter was facilitated by extensive chromosome length polymorphism in both strains. When crossing the two haploid strains S. cerevisiae 41 and S. diastaticus ATCC 28339 , two different types of hybrids occurred. Both types showed complete addition of both parental genomes, one a-status and the other -status. The -status could be explained by assuming a transient premutational lesion in MAT . Usually lesions are repaired after a mating event and the -mating type is restored. When crossing a diploid S. diastaticus strain, isogenic to the one previously mentioned, with the haploid S. cerevisiae strain, three different types of hybrids could be distinguished regarding their mating-types. It was possible to prove that the haploid S. diastaticus strain ATCC 28339 is disomic and the diploid hybrid, named 41ATCC-b, is trisomic for chromosome I. This could be shown by means of electrophoretic karyotyping of the hybrid and of the four single-spore cultures from one ascus of the hybrid.