Deacidification of grape juice with derepressed mutants of the yeast Hansenula anomala

Summary Transport and utilization of malic acid by the yeast Hansenula anomala are subject to glucose repression. Derepressed diploid mutant strains were obtained by hybridization of derepressed haploid mutant strains of opposite mating type. Six diploid mutant strains displayed derepressed behaviour with respect to malic acid utilization in the presence of glucose up to 30% (w/v). Three of these diploid mutant strains, as compared with the parent strain, were able to degrade completely malic acid in grape juice without fermenting the sugars. In addition, using one diploid mutant strain together with a strain of the wine yeast Saccharomyces cerevisiae, it was possible to carry out a mixedmicrovinification in which deacidification occurred simultaneously with alcoholic fermentation.     

Improvement in citric acid production by haploidization of Aspergillus niger diploid strains

Segregation of diploid strains by a haploidizing agent was used to improve citric acid producing strains of Aspergillus niger. Stable diploid strains were obtained via protoplast fusion between two citric acid-producing strains from different genealogies, one for shaking culture and the other for solid culture. Diploid strains were treated by benomyl as a haploidizing agent, and many segregants were obtained. Prototrophic segregants were selected and their haploidy was confirmed by their conidial size and DNA contents. The prototrophic segregants were very variable in their citric acid productivities, some of them better either in shaking culture or in solid culture than both the parental strains. The presence of methanol stimulated citric acid production by the parental and the diploid strains. However, all prototrophic segregants derived from one diploid strain had higher productivities in solid cultures without methanol than in those with methanol.     

Yeast ribosomal proteins: XIII. Saccharomyces cerevisiae YL8A gene, interrupted with two introns, encodes a homolog of mammalian L7.

We isolated and sequenced a gene, YL8A, encoding ribosomal protein YL8 of Saccharomyces cerevisiae. It is one of the two duplicated genes encoding YL8 and is located on chromosome VII while the other is on chromosome XVI. The haploid strains carrying disrupted YL8A grew more slowly than the parent strain. The open reading frame is interrupted with two introns. The predicted amino acid sequence reveals that yeast YL8 is a homolog of mammalian ribosomal protein L7, E.coli L30 and others.     

Efficient production of ethanol from raw starch by a mated diploid Saccharomyces cerevisiae with integrated α-amylase and glucoamylase genes

The goal of this research was to construct a stable and efficient process for the production of ethanol from raw starch, using a recombinant Saccharomyces cerevisiae, which is productive even under conditions such as non-selection or long-term operation. Three recombinant yeast strains were used, two haploid strains (MT8-1SS and NBRC1440SS) and one diploid strain (MN8140SS). The recombinant strains were constructed by integrating the glucoamylase gene from Rhizopus oryzae fused with the 3′-half of the α-agglutinin gene as the anchor protein, and the α-amylase gene from Streptococcus bovis, respectively, into their chromosomal DNA by homologous recombination. The diploid strain MN8140SS was constructed by mating these opposite types of integrant haploid strains in order to enhance the expression of integrated amylase genes. The diploid strain had the highest ethanol productivity and reusability during fermentation from raw starch. Moreover, the ethanol production rate of the integrant diploid strain was maintained when batch fermentation was repeated three times (0.67, 0.60, and 0.67 g/l/h in each batch). These results clearly show that a diploid strain developed by mating two integrant haploid strains is useful for the establishment of an efficient ethanol production process.     

Induction and Characterization of Artificial Diploids from the Haploid Yeast Torulaspora delbrueckii

The yeast Torulaspora delbrueckii, which propagates as a haploid, was made into a diploid by treatment with dimethyl sulfoxide (DMSO) on the regeneration of protoplasts. The diploid state was stably inherited; the cell volume was three times that of the parent strain and the cellular DNA content was two times that of the parental strain. No essential difference was found between diploids induced by DMSO and those formed through intraspecific protoplast fusion. The diploid strains sporulated fairly well, with their cells converting directly into asci. Random spore analysis revealed that diploids induced through protoplast fusion gave rise to auxotrophic segregants (haploids) with the parental genetic marker or to segregants formed by recombination, while diploids induced by DMSO from a doubly auxotrophic parent gave rise to no recombinant, indicating that it was chromosomally homoallelic in nature. The magnesium level in the protoplast regeneration medium was found to be an important factor for inducing diploid formation. At 0.2 mM magnesium diploids appeared even in the absence of DMSO, while at 2 mM magnesium diploids never appeared unless DMSO was added to the regeneration medium. Evidence is provided that the diploids induced by DMSO or a low magnesium level are due to direct diploidization but not protoplast fusion. UV light irradiation of intact cells (without protoplasts), 10% of which survived, also produced diploids among this surviving population. From these results we conclude that the perturbation of protoplast regeneration or of cell division by the treatments mentioned above somehow induced direct diploidization of T. delbrueckii.     

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.     

Properties of heterozygous diploids between strains ofPenicillium chrysogenum selected for high penicillin yield

Three strains ofPenicillium chrysogenum selected for high penicillin yield and of independent lineage were marked with suitable genetical characters prior to the synthesis of several heterozygous diploids. These parental strains had domestic codes, C, D and Y. Two diploids, between differently labelled mutants of strain C and Y, produced similar amounts of penicillin to strain C, which was less than that produced by strain Y. Previous work had indicated that genes responsible for increased penicillin yield were recessive and the present results suggested that such genes in strains C and Y were allelic, apart from the presence of one or more additional recessive mutations leading to greater penicillin production in the higher yielding parent. Three diploids made between mutants of strains D and Y were lower in penicillin yield than either original parent and only in the case of one diploid compared with one of the parental strains was this difference not significant. In strains D and Y, therefore, there may have been some recessive genes concerned with increasing penicillin yield which were non-allelic. However, no first order segregants arising spontaneously or subsequent to X-ray treatment produced higher levels of penicillin than the better yielding original parent in any cross.     

Citric acid production by the diploid strains of Aspergillus niger obtained by protoplast fusion

Summary Diploid strains were obtained following protoplast fusion between two citric acid producers of Aspergillus niger, one for the solid culture and the other for the shaking culture. In the shaking culture, all the diploid strains exhibited lower productivities than one parental strain. However, in the solid culture, some diploid strains exhibited higher productivities than either parental strain; the best diploid strain produced 1.2 times as much citric acid as the parental strain in solid culture.     

Stress tolerance in doughs of Saccharomyces cerevisiae trehalase mutants derived from commercial Baker's yeast.

Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (Deltanth1), acid trehalase mutants (Deltaath1), and double mutants (Deltanth1 ath1) by using commercial baker's yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The Deltanth1 and Deltaath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Deltanth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the trehalase mutants may make these strains useful in frozen dough.     

Isolation of Auxotrophic Mutants of Diploid Industrial Yeast Strains after UV Mutagenesis

Auxotrophic mutants of the yeast Saccharomyces cerevisiae are usually isolated in haploid strains because the isolation of recessive mutations in diploids is thought to be difficult due to the presence of two sets of genes. We show here that auxotrophic mutants of diploid industrial sake yeast strains were routinely obtained by a standard mutant selection procedure following UV mutagenesis. We isolated His⁻, Met⁻, Lys⁻, Trp⁻, Leu⁻, Arg⁻, and Ura⁻ auxotrophic mutants of five sake strains, Kyokai no. 7, no. 9, no. 10, no. 701, and no. 901, by screening only 1,700 to 3,400 colonies from each treated strain. Wild-type alleles were cloned and used as markers for transformation. With HIS3 as a selectable marker, the yeast TDH3 overexpression promoter was inserted upstream of ATF1, encoding alcohol acetyltransferase, by one-step gene replacement in a his3 mutant of Kyokai no. 7. The resulting strain contained exclusively yeast DNA, making it acceptable for commercial use, and produced a larger amount of isoamyl acetate, a banana-like flavor. We argue that the generally recognized difficulty of isolating auxotrophic mutants of diploid industrial yeast strains is misleading and that genetic techniques used for haploid laboratory strains are applicable for this purpose.     

Studies of the inheritance of virulence in the entomopathogenic fungus Metarhizium anisopliae

A forced heterocaryon was established between two auxotrophic conidial color mutants of Metarhizium anisopliae. From the heterocaryon, a prototrophic somatic diploid was selected which, in turn, yielded somatic segregants. The virulence of the original mutants, the somatic diploid, and the somatic segregants was evaluated on three species of mosquitoes as well as on Ostrinia nubilalis larvae. The virulence of the somatic diploid was comparable to that of the wild-type parental strain while the auxotrophic somatic segregants exhibited virulence approximately equal to that of the auxotrophic components of the heterocaryon. Putative somatic diploids were obtained between morphological mutants of the two species varieties (M. anisopliae var. minor and var. major). The presumptive diploids were avirulent for the insect species to which the parental strains exhibited virulence.     

Overexpression of bacterial xylose isomerase and yeast host xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha

The thermotolerant methylotrophic yeast Hansenula polymorpha is able to ferment xylose to ethanol. To improve characteristics of xylose fermentation, the recombinant strain Delta xyl1 Delta xyl2-ADelta xyl2-B, with deletions of genes encoding first enzymes of xylose utilization (NAD(P)H-dependent xylose reductase and NAD-dependent xylitol dehydrogenases, respectively), was constructed and used as a recipient for co-overexpression of the Escherichia coli xylA gene coding for xylose isomerase and endogenous XYL3 gene coding for xylulokinase. The expression of both genes was driven by the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase promoter. Xylose isomerase activities of obtained transformants amounted to approximately 80% of that of the bacterial host strain. Xylulokinase activities of the transformants increased twofold when compared with the parental strain. The recombinant strains displayed improved ethanol production during the fermentation of xylose.     

hREV3 is essential for error-prone translesion synthesis past UV or benzo[a]pyrene diol epoxide-induced DNA lesions in human fibroblasts

In S. cerevisiae, the REV3 gene, encoding the catalytic subunit of polymerase zeta, is involved in translesion synthesis and required for the production of mutations induced by ultraviolet radiation (UV) photoproducts and other DNA fork-blocking lesions, and for the majority of spontaneous mutations. To determine whether hREV3, the human homolog of yeast REV3, is similarly involved in error-prone translesion synthesis past UV photoproducts and other lesions that block DNA replication, an hREV3 antisense construct under the control of the TetP promoter was transfected into an infinite life span human fibroblast cell strain that expresses a high level of tTAk, the activator of that promoter. Three transfectant strains expressing high levels of hREV3 antisense RNA were identified and compared with their parental cell strain for sensitivity to the cytotoxic and mutagenic effects of UV. The three hREV3 antisense-expressing cell strains were not more sensitive than the parental strain to the cytotoxic effect of UV, but the frequency of mutants induced by UV in their HPRT gene was significantly reduced, i.e. to 14% that of the parent. Two of these hREV3 antisense-expressing cell strains were compared with the parental strain for sensitivity to (±)-7β,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). They were not more sensitive than the parent strain to the cytotoxic effect of BPDE, but the frequency of mutants induced was significantly reduced, i.e. in one strain, to 17% that of the parent, and in the other, to 24%. DNA sequencing showed that the kinds of mutations induced by BPDE in the parental and the derivative strains did not differ and were similar to those found previously with finite life span human fibroblasts. The data strongly support the hypothesis that hRev3 plays a critical role in the induction of mutations by UV or BPDE. Because the level of hRev3 protein in human fibroblasts is below the level of antibody detection, it was not possible to demonstrate that the decrease in mutagenesis reflected decreased hRev3 protein. However, the conclusion is supported by the fact that in a similar study with a strain expressing a high level of antisense hREV1, a very similar result was obtained, i.e. UV or BPDE mutagenesis was virtually eliminated.     

Ploidy reduction usingp-Fluorophenylalanine of fusion products ofSaccharomyces cerevisiae

Fusion of yeast protoplasts was induced in the presence of polyethylene glycol and Ca++ ions. Two auxotrophic complementingSaccharomyces cerevisiae mutant strains were used in fusion experiments. One diploid and several polyploid fusion products were selected by complementation in minimal medium. The assessment of ploidy has been based on the DNA content of the parental cells and fusion products, assayed with the diphenylamine method. Treating the fusion product cells withp-Fluorophenylalanine (p-FPA), parentalhis andleu markers could not be recovered. Instead, a strong reduction of polyploid fusion product cell DNA content was evident. The analysis of meiotic products after hybridizing one fusion product with a prototrophicSaccharomyces cerevisiae standard strain led to the recovery of thehis parental marker. Preliminary evidence thatp-Fluorophenylalanine could be used as a diploidizing agent towards polyploid strains ofSaccharomyces cerevisiae is reported.     

基因组改组技术选育耐酸性琥珀酸放线杆菌

以琥珀酸产生菌Actinobacillus succinogenes CGMCC 1593为出发菌,分别经过紫外线-甲基磺酸乙酯(UV-EMS)和紫外线-硫酸二乙酯(UV-DES)诱变处理,得到7株耐酸性有所提高的突变株.以此作为候选菌库,经3轮原生质体递进融合,筛选获得4株可以在pH 5.6下生长的改组菌株.其中改组菌株F3-21在pH 5.6的完全液体培养基中生长的OD值是原始菌的7倍,在pH 5.2条件下仍能生长;其摇瓶发酵48h琥珀酸产量较原始菌株提高48%.在5L发酵罐中进行分批发酵,当控制pH在较低值(5.6～6.0)时,F3-21厌氧发酵48h积累琥珀酸38.1g/L,较出发菌株提高了45%;当控制pH在6.5～7.0时,F3-21厌氧发酵32h积累琥珀酸40.7g/L.F3-21在5L发酵罐中进行补料分批发酵,厌氧发酵72h,产琥珀酸达67.4g/L.结果说明基因组改组技术能够改进琥珀酸放线菌的耐酸性能及其琥珀酸的产量.     

Inherent G418-resistance in hybridization of industrial yeasts

Eight strains of sake yeast exhibited inherent-resistance to 100 μg/ml of Geneticin (G418). Fourteen wine yeasts and 1 shochu yeast (Saccharomyces cerevisiae) and 1 miso yeast (Zygosaccharomyces rouxii) were inherent G418-sensitive. The petites converted from inherent G418-resistants by treatment with ethidium bromide retained G418-resistance (ϱ⁻ G418R), and thus were hybridized by electrofusion with the wine yeast W3 (ϱ⁺ G418S, wild type). A lag phase of 12–18 h was required prior to administration of the drug in glycerol medium when selecting G418-resistant hybridization products. Colonies were formed in the regeneration medium at a frequency of about 1 × 10⁻⁵ per used protoplasts. No growth of any parental strain (10⁶/_~10⁷ protoplasts) separately subjected to electrofusion and regeneration was observed. The hybridization products were G418-resistant “grande” strains (ϱ⁻ G418R) in which the genetic traits of parental strains had been complemented. Uninucleate cells (DAPI staining) of the hybridization products showed CHEF electrophoretic karyotypes similar to that of wine yeast, but possessed a single chromosome (approx. 320 kb) presumably from sake yeast.     

Isolation of sulfite reductase variants of a commercial wine yeast with significantly reduced hydrogen sulfide production

The production of hydrogen sulfide (H₂S) during fermentation is a common and significant problem in the global wine industry as it imparts undesirable off-flavors at low concentrations. The yeast Saccharomyces cerevisiae plays a crucial role in the production of volatile sulfur compounds in wine. In this respect, H₂S is a necessary intermediate in the assimilation of sulfur by yeast through the sulfate reduction sequence with the key enzyme being sulfite reductase. In this study, we used a classical mutagenesis method to develop and isolate a series of strains, derived from a commercial diploid wine yeast (PDM), which showed a drastic reduction in H₂S production in both synthetic and grape juice fermentations. Specific mutations in the MET10 and MET5 genes, which encode the catalytic α- and β-subunits of the sulfite reductase enzyme, respectively, were identified in six of the isolated strains. Fermentations with these strains indicated that, in comparison with the parent strain, H₂S production was reduced by 50–99%, depending on the strain. Further analysis of the wines made with the selected strains indicated that basic chemical parameters were similar to the parent strain except for total sulfite production, which was much higher in some of the mutant strains.     

Allelic and Ectopic Interactions in Recombination-Defective Yeast Strains

Ectopic recombination in the yeast Saccharomyces cerevisiae has been investigated by examining the effects of mutations known to alter allelic recombination frequencies. A haploid yeast strain disomic for chromosome III was constructed in which allelic recombination can be monitored using leu2 heteroalleles on chromosome III and ectopic recombination can be monitored using ura3 heteroalleles on chromosomes V and II. This strain contains the spo13-1 mutation which permits haploid strains to successfully complete meiosis and which rescues many recombination-defective mutants from the associated meiotic lethality. Mutations in the genes RAD50, SPO11 and HOP1 were introduced individually into this disomic strain using transformation procedures. Mitotic and meiotic comparisons of each mutant strain with the wild-type parental strain has shown that the mutation in question has comparable effects on ectopic and allelic recombination. Similar results have been obtained using diploid strains constructed by mating MATa and MAT alpha haploid derivatives of each of the disomic strains. These data demonstrate that ectopic and allelic recombination are affected by the same gene products and suggest that the two types of recombination are mechanistically similar. In addition, the comparison of disomic and diploid strains indicates that the presence of a chromosome pairing partner during meiosis does not affect the frequency of ectopic recombination events involving nonhomologous chromosomes.     

Disruption of TRI101, the Gene Encoding Trichothecene 3-O-Acetyltransferase, from Fusarium sporotrichioides

We screened a Fusarium sporotrichioides NRRL 3299 cDNA expression library in a toxin-sensitive Saccharomyces cerevisiae strain lacking a functional PDR5 gene. Fourteen yeast transformants were identified as resistant to the trichothecene 4,15-diacetoxyscirpenol, and each carried a cDNA encoding the trichothecene 3-O-acetyltransferase that is the F. sporotrichioides homolog of the Fusarium graminearum TRI101 gene. Mutants of F. sporotrichioides NRRL 3299 produced by disruption of TRI101 were altered in their abilities to synthesize T-2 toxin and accumulated isotrichodermol and small amounts of 3,15-didecalonectrin and 3-decalonectrin, trichothecenes that are not observed in cultures of the parent strain. Our results indicate that TRI101 converts isotrichodermol to isotrichodermin and is required for the biosynthesis of T-2 toxin.     

Mutations of Francisella novicida that Alter the Mechanism of Its Phagocytosis by Murine Macrophages

Infection with the bacterial pathogen Francisella tularensis tularensis (F. tularensis) causes tularemia, a serious and debilitating disease. Francisella tularensis novicida strain U112 (abbreviated F. novicida), which is closely related to F. tularensis, is pathogenic for mice but not for man, making it an ideal model system for tularemia. Intracellular pathogens like Francisella inhibit the innate immune response, thereby avoiding immune recognition and death of the infected cell. Because activation of inflammatory pathways may lead to cell death, we reasoned that we could identify bacterial genes involved in inhibiting inflammation by isolating mutants that killed infected cells faster than the wild-type parent. We screened a comprehensive transposon library of F. novicida for mutant strains that increased the rate of cell death following infection in J774 macrophage-like cells, as compared to wild-type F. novicida. Mutations in 28 genes were identified as being hypercytotoxic to both J774 and primary macrophages of which 12 were less virulent in a mouse infection model. Surprisingly, we found that F. novicida with mutations in four genes (lpcC, manB, manC and kdtA) were taken up by and killed macrophages at a much higher rate than the parent strain, even upon treatment with cytochalasin D (cytD), a classic inhibitor of macrophage phagocytosis. At least 10-fold more mutant bacteria were internalized by macrophages as compared to the parent strain if the bacteria were first fixed with formaldehyde, suggesting a surface structure is required for the high phagocytosis rate. However, bacteria were required to be viable for macrophage toxicity. The four mutant strains do not make a complete LPS but instead have an exposed lipid A. Interestingly, other mutations that result in an exposed LPS core were not taken up at increased frequency nor did they kill host cells more than the parent. These results suggest an alternative, more efficient macrophage uptake mechanism for Francisella that requires exposure of a specific bacterial surface structure(s) but results in increased cell death following internalization of live bacteria.