Intergeneric hybrids of Saccharomyces cerevisiae and Zygosaccharomyces fermentati obtained by protoplast fusion.

To obtain strains that are able to efficiently produce ethanol from different carbohydrates, mainly cellulose hydrolysates, several species of the genus Candida and a Zygosaccharomyces fermentati strain were examined for their ability to utilize cellobiose and produce ethanol, as well as for their thermotolerance and the possibility of genetic manipulation. Candida obtusa and Zygosaccharomyces fermentati tolerated the maximal temperature for growth, possessed the highest cellobiase activity, and offered the possibility of genetic manipulation, although neither of them proved to be a good producer of ethanol. Intergeneric hybrids of Saccharomyces cerevisiae and Z. fermentati were obtained after protoplast fusion. They were selected as prototrophic strains, after isolation of auxotrophic mutants from Z. fermentati and fusion with an S. cerevisiae strain which was also auxotrophic. The hybrids, which appeared at a frequency of 2 X 10(-7), presented characteristics of both parents, such as resistance to certain drugs and the ability to grow with either cellobiose or lactic acid as the sole carbon source; they were very stable, even under nonselective conditions. These hybrids may have important industrial applications as good fermenting strains.     

Characterization of hybrids obtained by protoplast fusion,between Pachysolen tannophilus and Saccharomyces cerevisiae

The genes for utilization of xylose were transferred from Pachysolen tannophilus to Saccharomyces cerevisiae. The hybrids resembled the S. cerevisiae parent morphologically and in sugar assimilation. Pulsed field gel electrophoresis showed that the chromosome banding pattern was intermediate between the two parental species. Correspondence to: H. Heluane     

Characterization of intergeneric hybrids obtained by protoplast fusion between Phaffia rhodozyma, Cryptoococcus laurentii and Saccharomyces cerevisiae

Protoplast fusion was used to obtain hybrids between Phaffia rhodozymaand Cryptococcus laurentii or Saccharomyces cerevisiae(petite form). Hybrids were obtained which produced the carotenoids b-carotene, phytoene and phytofluene, and were respiratory-competent and prototrophic.     

Multiple transformation of Saccharomyces cerevisiae by protoplast fusion

A technique for the multiple transformation of yeast by protoplast fusion is described. This involved the PEG-induced fusion of protoplasts from cells which had been treated with chromosome-fragmenting agents (in this case cupferron and hydroxylamine) with protoplasts of triply auxotrophic cells. The recovery of transformants was increased significantly if one of the amino acid requirements of the recipient strain was included in the selection medium. Transformants isolated on supplemented media remained auxotrophic for that requirement. Prototrophic, uninucleate transformants had a DNA content and cellular volume similar to that of the parental strains. Possible mechanisms of gene transfer are discussed. This technique offers the possibility of transferring desirable characteristics from one yeast strain to another without altering the ploidy level of the recipient strain.     

Intergeneric ethanol producing hybrids of thermotolerant Kluyveromyces and non-thermotolerant Saccharomyces cerevisiae

Intergeneric hybrids of an Arg auxotroph of Kluyveromyces marxianus able to grow up to 52¡C and Saccharomyces cerevisiae capable of growth up to 40¡C, have been constructed by protoplasmic fusion. The fusants resembled the former except that they were prototrophic and some produced ethanol in excess of 6% (v/v) both at 30 and 45¡C, an attribute otherwise lacking in the parents. The hybrids were stable during mitotic and meiotic growth. Genetic evidence leading to the retrieval of orthodox arg at a frequency of 6% from the prototrophic hybrids, confirmed their genuineness and the presence of S. cerevisiae-specific ARG in an integrative manner. The integration seemed to have occurred at a locus ~12 cM away from the orthodox arg which on the other side was found to be linked to MET. The order of three genes was, therefore, speculated to be either MET-arg-ARG or ARG-arg-MET.     

Interspecific protoplast fusion of the Baker's yeast Saccharomyces cerevisiae and Saccharomyces diastaticus

Summary The protoplast fusion technique provides a useful method for improving industrial yeasts and agglutinant agents like polyethylene glycol (PEG) MW 4000 and Ca⁺⁺ ions are widely used to stimulate the fusion process. Commercial Baker's yeast Saccharomyces cerevisiae and Saccharomyces diastaticus were selected as parental strains for somatic fusion. The Saccharomyces diastaticus carried a spontaneous petite mutation and could not metabolize starch unlike respiratory competent Saccharomyces diastaticus from which it was derived, that readily could.A medium containing soluble starch as a carbon source and 3 % agar was used as fusion products selection medium. Respiratory competent fusion products were capable of using dextrins and starch as carbon sources.     

Intergeneric gene transfer mediated by plant protoplast fusion

Summary In attempts at somatic transfer of plant genomes of reduced size, X-irradiated leaf protoplasts of parsley (Petroselinum hortense, 2n=22) were fused with cell culture protoplasts of a nuclear albino mutant of carrot (Daucus carota, 2n=18). Introduction of genes from the irradiated parsley nuclei into the carrot genome was shown by the correction of the albino defect and by the appearance of parsley isoenzymes in selected green tissues and plants. The cytological studies provided information on significant deviation from the amphidiploid chromosome number. The high frequency of cells with 2n=19, 2n=38 and regeneration of plants with 2n=19 chromosomes can indicate that the elimination of parsley chromosomes is incomplete. A correlation was found between the lethality of selected tissues and differentiated or undifferentiated stages of the cells.     

High-efficiency transformation of Saccharomyces cerevisiae cells by bacterial minicell protoplast fusion.

After a new transformation procedure, 10% of Saccharomyces cerevisiae cells were found to contain transforming DNA sequences. We used direct transfer of plasmid molecules by fusing bacterial minicell protoplasts to yeast protoplasts. Since the procedure significantly reduces the toxic effect of procaryotic protoplasm on the eucaryotic organism, it might be generally applicable in other systems in which transformation is inefficient or impossible.     

Intergeneric hybridization betweenMonascus anka andAspergillus oryzae by protoplast fusion

Summary To breed industrially useful strains of a slow-growing, red-pigment-producing strain ofMonascus anka, protoplasts ofM. anka MAK1 (arg) andAspergillus oryzae AOK1 (met, thr) were fused. A mixture of protoplasts prepared from mycelia ofM. anka MAK1 treated with 2% Usukizyme and ofA. oryzae AOK1 treated with 2% Usukizyme and 0.2% NovoZym 234 was incubated with 30% (w/v) polyethylene glycol no. 6000. Heterokaryon fusants complementing the auxotrophies of both mutants were isolated on minimal medium, but segregated into red (MAK1) and white (AOK1) sectors after being cultured on a complete medium. After irradiation with UV light, the fusants gave stable heterozygous diploids that formed long white hyphae. These diploids, which had twice as much DNA in the nucleus as their parents, grew more rapidly than the parent strain YZT1, and produced ethanol earlier than the parents. Production of amylase, protease, and kojic acid by the fusants was intermediate in amount between that of the two parents.     

Characterisation of osmotolerant hybrids obtained by fusion between protoplasts of Saccharomyces cerevisiae and heat treated protoplasts of Torulaspora delbrueckii

Saccharomyces cerevisiae was fused with heat-treated protoplasts of an osmotolerant yeast, Torulaspora delbrueckii, to obtain hybrids having increased tolerance to increased glucose concentrations (up to 700 gl–1). The production of glycerol and arabitol by the hybrids was within the range of those of the parental strains, but the production of ethanol was higher.     

Intergeneric protoplast fusion in lactic acid bacteria

Abstract Crystals from Bacillus thuringiensis var. israelensis appeared to contain three major proteins of M _r 230 000, 130 000 and 28 000. These proteins were solubilized from the crystals by incubation in 10 mM DTT, pH 9.5, and purified by sucrose gradient centrifugation. The M _r 230 000 and 130 000 crystal proteins showed mosquitocidal properties, whereas the M _r 28 000 crystal protein contained haemolytic activity. Immobilization of these proteins on latex beads did not alter these properties. Partial proteolytic degradation showed that the M _r 130 000 and 28 000 proteins are structurally different.     

The effect of a-factor on hybrid formation by protoplast fusion in Saccharomyces cerevisiae

Abstract a_˜-Factor, unlike α-factor, does not significantly enhance hybrid formation by protoplast fusion in the yeast Saccharomyces cerevisiae . When Mat α cells are treated with a-factor prior to being proto-plasted and fused, the frequency of hybrid formation is only slightly increased over unarrested controls.     

Glycerol and arabitol production by an intergeneric hybrid,PB2, obtained by protoplast fusion between Saccharomyces cerevisiae and Torulaspora delbrueckii

An intergeneric osmotolerant hybrid yeast, PB2, was used together with the parental strains to study glycerol and arabitol production in batch culture. This fusion product was previously obtained by protoplast fusion between Torulaspora delbrueckii and Saccharomyces cerevisiae. Polyols and biomass production were determined in batch culture under aerobic conditions. Under the conditions tested, using PB2 hybrid and both parental strains, the best results were obtained with the hybrid. Arabitol reached a final concentration of 70 g/l and glycerol was increased to up to 50 g/l. Electronic Publication     

Alpha-factor enhancement of hybrid formation by protoplast fusion in the yeast Saccharomyces cerevisiae

Two a strains of Saccharomyces cerevisiae, carrying complementary genetic markers, were arrested for 3 h with alpha-factor. These were then protoplasted, prior to fusion using polyethylene glycol. The number of viable fusion products was enhanced by a factor of 20 as compared with unarrested controls.     

Use of protoplast fusion to introduce methionine overproduction into Saccharomyces cerevisiae

Summary dl-Ethionine-resistant mutants of Saccharomyces uvarum ATCC 26602 were found to overproduce exogenous l-methionine. dl-Ethionine-resistant mutant ER 108, carrying a mutation to chloramphenicol resistance was converted to petite form, and protoplasts obtained from it were fused with protoplasts from antibiotic-sensitive S. cerevisiae X2928 carrying six auxotrophies. The resulting fusants maintained four auxotrophies and were capable of overproducing l-methionine. These fusants were stable after ten passages on complete medium.