Laser mutagenesis of protoplasts of Penicillium sp. PT95 for the enhancement of carotenoid yield

Aims: To isolate the protoplasts from Penicillium sp. PT95 and carry out laser mutagenesis to attain high-yield mutant strain for carotenoid production. Methods and Results: The mycelial pellets of PT95 strain were digested with the lytic enzyme for 3 h in order to attain protoplasts. The prepared protoplasts were irradiated using helium neon (He–Ne) laser. Among all regenerated colonies isolated from irradiated protoplasts, five colonies proved to be able to form sclerotia. The five colonies were named as strains L01, L02, L03, L04 and L05, respectively. Whereas, among all regenerated colonies isolated from no-irradiated protoplasts, no colonies were found to form sclerotia. Strains L01, L02, L03, L04 and L05 showed higher carotenoid yield than the original strain in Czapek’s agar medium. Strain L05 gave the highest pigment yield of 381 μg per plate, which was 2·54 times higher than that of original strain. Conclusions: These results suggest that PT95 strain may be mutagenized using laser-irradiation to obtain higher-yield mutant strains for carotenoid production. Significance and Impact of the Study: These data prompted us to consider that several attempts should be made to improve carotenoid production in PT95 by strain selection using classical screening and mutagenesis techniques.     

Characterization of glucose transport inSchizosaccharomyces pombe

Conclusions GHT1 was isolated as suppressor ofd-glucose uptake deficiency ofS. pombe mutant YGS-5. The correspondingS. pombe DNA encodes a putative protein with significant amino acid sequence identity to theS. cerevisiae HXT transporters. Heterologous expression ofGHT1 inS. cerevisiae hxt mutant RE700A (strain HLY709) enabled the mutant to grow ond-glucose as the sole carbon source. HLY709 cells take up hexoses with similar specificity toS. pombe wild strain and accumulate the non-metabolizable analogues of glucose (2DG and 6DG) intracellularly, thus matchingS. pombe wild strain. Southern blot analysis revealed the existence of other putative glucose transporters inS. pombe and the search for related transporter genes inS. pombe genome is in progress.     

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.     

Preparation and regeneration of protoplasts and spheroplasts for fusion and transformation ofSchizosaccharomyces pombe

Preparation and regeneration of protoplasts is essential for somatic hybridization and transformation of yeasts. We present conditions that were found to be optimal for preparing and regeneratingSchizosaccharomyces pombe protoplasts for cell fusion. In contrast to these conditions, genetic transformation ofS. pombe requires spheroplasts that are osmotically sensitive, but still have some wall material attached to the cell. The main finding were as follows: (a) For protoplast formation with Novozym SP234, 0.9M sorbitol was found to be the optimal osmotic milieu and -mercaptoethanol is not necessary. (b) Embedding in soft agar yields considerably better regeneration frequencies than direct plating. (c) Cell fusion is optimal when both fusion partners are fully protoplasted, although considerable fusion occurs between spheroplasted cells as well. (d)Schizosaccharomyces pombe transformation frequencies are much higher with spheroplasts than with protoplasts. Inclusion of -mercaptoethanol did not enhance transformation frequency.     

Transfer of isolated nuclei into protoplasts ofSaccharomyces cerevisiae

Cell nuclei were prepared from protoplasts of an adenine-requiring strain ofSaccharomyces cerevisiae, then purified in a discontinuous sucrose gradient, and applied to protoplasts of a recipient strain auxotrophic for uracil, leucine, and histidine. The transfer of the isolated nuclei into protoplasts was induced with polyethylene glycol. The main products of nuclear transfer in young complemented colonies were heterokaryons giving rise to parental type spontaneuos segregants on nutritionally complete medium. After several passages in minimal medium, however, the prototrophic colonies consisted exclusively of stable heterozygous diploid cells.     

The gld1 + gene encoding glycerol dehydrogenase is required for glycerol metabolism in Schizosaccharomyces pombe

The budding yeast Saccharomyces cerevisiae is able to utilize glycerol as the sole carbon source via two pathways (glycerol 3-phosphate pathway and dihydroxyacetone [DHA] pathway). In contrast, the fission yeast Schizosaccharomyces pombe does not grow on media containing glycerol as the sole carbon source. However, in the presence of other carbon sources such as galactose and ethanol, S. pombe could assimilate glycerol and glycerol was preferentially utilized over ethanol and galactose. No equivalent of S. cerevisiae Gcy1/glycerol dehydrogenase has been identified in S. pombe. However, we identified a gene in S. pombe, SPAC13F5.03c (gld1 ⁺), that is homologous to bacterial glycerol dehydrogenase. Deletion of gld1 caused a reduction in glycerol dehydrogenase activity and prevented glycerol assimilation. The gld1Δ cells grew on 50 mM DHA as the sole carbon source, indicating that the glycerol dehydrogenase encoded by gld1 ⁺ is essential for glycerol assimilation in S. pombe. Strains of S. pombe deleted for dak1 ⁺ and dak2 ⁺ encoding DHA kinases could not grow on glycerol and showed sensitivity to a higher concentration of DHA. The dak1Δ strain showed a more severe reduction of growth on glycerol and DHA than the dak2Δ strain because the expression of dak1 ⁺ mRNA was higher than that of dak2 ⁺. In wild-type S. pombe, expression of the gld1 ⁺, dak1 ⁺, and dak2 ⁺ genes was repressed at a high concentration of glucose and was derepressed during glucose starvation. We found that gld1 ⁺ was regulated by glucose repression and that it was derepressed in scr1Δ and tup12Δ strains.     

Regeneration of plants from protoplasts of Capsella bursa-pastoris and somatic hybridization with rapid cycling Brassica oleracea

Fertile rooted plantlets were recovered from leaf mesophyll protoplasts of Capsella bursa-pastoris. Protoplasts cultured over a feeder layer of Brassica napus cells produced 221 colonies, 7 of which regenerated multiple plantlets. The nuclear DNA content of most regenerates varied from 0.89 to 1.0 pg/nucleus, close to the value for seed-grown C. bursa-pastoris (0.94±0.03 pg/nucleus). Two regenerants had a tetraploid DNA content (1.8– 2.0 pg). Plants with a DNA content close to Capsella produced seeds, both in vitro and in soil. Intertribal somatic hybrids were obtained by polyethylene glycol-mediated fusion of untreated C. bursa-pastoris protoplasts with iodoacetate-treated protoplasts of rapid-cycling B. oleracea. Plants were confirmed as somatic hybrids by isozyme and RAPD analysis. The nuclear DNA content of the hybrids ranged from 3.2 to 6.4 pg, higher than the sum of the parental genomes. One of two hybrids tested was resistant to Alternaria brassicicola, like the Capsella fusion partner. Hybrids rooted easily and produced sterile flowers when transplanted to soil. Received: 13 April 1998 / Revision received: 25 August 1998 / Accepted: 31 August 1998     

An improved method for protoplast formation and its application in the fusion of Rhodotorula rubra with Saccharomyces cerevisiae

Protoplasts from various strains of red-pigmented yeasts were generated at high frequency using improved procedures. The use of sulphur-containing amino acids and 2-deoxyglucose in growth media led to impaired cell wall synthesis and rendered cells very susceptible to treatment with mercapto-ethanol and various lytic enzymes. Use of individual lytic enzymes separately resulted in relatively low frequencies of protoplasts from most of the red yeasts examined, whilst use of -glucoronidase, Novozyme and Zymolyase in series markedly increased stable protoplast formation. The latter effects were shown to be strain specific. The ability to generate large numbers of red yeast protoplasts prompted the attempt to examine intergeneric fusion between auxotrophs of a strain of Saccharomyces cerevisiae and Rhodotorula rubra. Putative hybrids were selected as variously-pigmented prototrophic colonies growing on minimal medium and stabilised by subculturing on the latter medium. Unusual cream, orange and yellow hybrid colonies were generated, composed of cells of varying morphologies (chains, multibudded). The majority of stable hybrids contained one nucleus, although several heterokaryons were also observed. Some hybrids possessed the phenotypes of both parents: fusant wcat41 grew as rapidly as the S. cerevisiae parent but also contained an inducible phenylalanine ammonia-lyase (PAL) which appeared to be more active than that of the Rhodotorula parent.     

Isolation of a malachite green-degrading Pseudomonas sp. MDB-1 strain and cloning of the tmr2 gene

The release of malachite green, a commonly used triphenylmethane dye, into the environment is causing increasing concern due to its toxicity, mutagenicity, and carcinogenicity. A bacterial strain that could degrade malachite green was isolated from the water of an aquatic hatchery. It was identified as a Pseudomonas sp. based on the morphological, physiological, and biochemical characteristics, as well as the analysis of 16S rRNA gene sequence and designated as MDB-1. This strain was capable of degrading both malachite green and leucomalachite green, as well as other triphenylmethane dyes including Crystal Violet and Basic Fuchsin. The gene tmr2, encoding the triphenylmethane reductase from MDB-1, was cloned, sequenced and effectively expressed in E. coli. These results highlight the potential of this bacterium for the bioremediation of aquatic environments contaminated by malachite green.     

N-glycans are not required for the efficient degradation of the mutant Saccharomyces cerevisiae CPY* in Schizosaccharomyces pombe

In eukaryotic cells, aberrant proteins generated in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation (ERAD) pathway. Here, we report on the ERAD pathway of the fission yeast Schizosaccharomyces pombe. We constructed and expressed Saccharomyces cerevisiae wild-type CPY (ScCPY) and CPY-G255R mutant (ScCPY*) in S. pombe. While ScCPY was glycosylated and efficiently transported to the vacuoles in S. pombe, ScCPY* was retained in the ER and was not processed to the matured form in these cells. Cycloheximide chase experiments revealed that ScCPY* was rapidly degraded in S. pombe, and its degradation depended on Hrd1p and Ubc7p homologs. We also found that Mnl1p and Yos9p, proteins that are essential for ERAD in S. cerevisiae, were not required for ScCPY* degradation in S. pombe. Moreover, the null-glycosylation mutant of ScCPY, CPY*0000, was rapidly degraded by the ERAD pathway. These results suggested that N-linked oligosaccharides are not important for the recognition of luminal proteins for ERAD in S. pombe cells.     

The Hermes transposon of Musca domestica and its use as a mutagen of Schizosaccharomyces pombe

Transposon mutagenesis allows for the discovery and characterization of genes by creating mutations that can be easily mapped and sequenced. Moreover, this method allows for a relatively unbiased approach to isolating genes of interest. Recently, a system of transposon based mutagenesis for Schizosaccharomyces pombe became available. This mutagenesis relies on Hermes, a DNA transposon from the house fly that readily integrates into the chromosomes of S. pombe. The Hermes system is distinct from the retrotransposons of S. pombe because it efficiently integrates into open reading frames. To mutagenize S. pombe, cells are transformed with a plasmid that contains a drug resistance marker flanked by the terminal inverted repeats of Hermes. The Hermes transposase expressed from a second plasmid excises the resistance marker with the inverted repeats and inserts this DNA into chromosomal sites. After S. pombe with these two plasmids grow 25 generations, approximately 2% of the cells contain insertions. Of the cells with insertions, 68% contain single integration events. The protocols listed here provide the detailed information necessary to mutagenize a strain of interest, screen for specific phenotypes, and sequence the positions of insertion.     

Ferrichrome in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> – an iron transport and iron storage compound

Schizosaccharomyces pombe has been assumed not to produce siderophores. Nevertheless, the genomic sequence of this fission yeast revealed the presence of siderophore biosynthetic genes for hydroxamates. Applying a bioassay based on an Aspergillus nidulans strain deficient in siderophore biosynthesis, and using reversed-phase HPLC and mass spectrometry analysis, we demonstrate that S. pombe excretes and accumulates intracellularly the hydroxamate-type siderophore ferrichrome. Under iron-limiting conditions, the cellular ferrichrome pool was present in the desferri-form, while under iron-rich conditions, in the ferri-form. In contrast to S. pombe, hydroxamate-type siderophores could not be detected in two other yeast species, Saccharomyces cerevisiae and Candida albicans.     

First identification of an amber nonsense mutation in Schizosaccharomyces pombe: major differences in the efficiency of homologous versus heterologous yeast suppressor tRNA genes

Summary In Saccharomyces cerevisiae ochre and opal, as well as amber mutations are known, whereas in the fission yeast Schizosaccharomyces pombe no amber alleles have been described. We have characterized trp1-566, an amber allele in the trp1 locus of S. pombe. The identification of trp1-566 as an amber allele is based on the following results: (a) The nonsense allele can be converted to an ochre allele by nitrosoguanidine mutagenesis. (b) trp1-566 is suppressed by a bona fide S. pombe amber suppressor tRNA, supSI. The supSI gene was obtained by primer-directed in vitro mutagenesis of a tRNASer from S. pombe. Unexpectedly, an S. cerevisiae amber suppressor tRNASer, supR21, transformed into S. pombe, failed to suppress trp1-566. Northern analysis of S. pombe transformants, containing supRL1 or S. cerevisiae tRNA^Leu or tRNA^Tyr genes reveals that these genes are not transcribed in the fission yeast. As an additional tool for the analysis of nonsense mutations in S. pombe, we obtained by nitrosoguanidine mutagenesis two unlinked amber suppressor alleles, sup13 and sup14, which act on trp1-566.     

A variant of Saccharomyces cerevisiae pep4 strain with improved oligotrophic proliferation, cell survival and heterologous secretion of α-amylase

A variant of Saccharomyces cerevisiae pep4 strain 20B12, with improved oligotrophic proliferation, cell survival and secretion of heterologous mouse α-amylase, is described. Previously we reported a procedure to enrich NI transformants that are not inhibited by cytotoxic expression of hepatitis B virus surface antigen in the secretion pathway of the protease-A-deficient (pep4) strain. To use the NI cells as a host for heterologous expression, we tried to amend the introduced pYAS/12S vector and obtain a host strain, NI-C, with stable NI phenotype and trp1 marker restored. Southern analysis of genomic DNA of NI-C suggested that the original pYAS/12S was abnormally rearranged and not completely corrected. Further assay showed that the viability and mitotic ability of the NI-C strain were increased. While using the NI-C strain as host for plasmid transformation and heterologous expression of mouse α-amylase, we observed that transformed colonies grew more quickly and secreted more α-amylase than general yeast strains. A further test showed that the NI-C strain was able to use mouse α-amylase as a positive selection marker to form transformed colonies on nitrogen-starved plates that contain starch as the sole carbon source. The results imply that the NI-C variant is an improved pep4 strain that can be used for heterologous expression and for the development of new selective markers in the yeast transformation system. Received: 7 January 1998 / Received last revision: 7 September 1998 / Accepted: 11 October 1998     

Production and characterization of somatic hybrids between Solanum melongena L. and S. sisymbriifolium Lam.

Summary Protoplasts of 6-azauracil (AU) resistant cell lines of Solanum melongena L. were fused with protoplasts of S. sisymbriifolium Lam. to create somatic hybrids between these sexually-incompatible species. Following fusion, colonies were selected which were capable of growth in medium containing 1mM AU. These colonies were placed on medium containing zeatin which had been shown to stimulate anthocyanin production during shoot organogenesis in tissue explants of S. sisymbriifolium but not in S. melongena. A total of 37 anthocyanin-producing colonies were identified from which 26 hybrid plants were regenerated. The morphological traits intermediate to those of the parents included: flower colour, leaf shape, and trichome density. Cytogenetic analysis revealed that all hybrids were aneuploids but their chromosome numbers were close to the expected number of 48. Isozyme analysis revealed that nuclear genes of both parents were expressed in the hybrids. In addition, isoelectric focussing of the large subunit of ribulose 1,5-bisphosphate carboxylase (Rubisco) provided evidence that each hybrid expressed only the S. sisymbriifolium chloroplast genome. All hybrids regenerated thus far have been sterile.Contribution No. 787 Ottawa Research Station     

Inhibition of Protein Translocation in Permeabilized Cells of Schizosaccharomyces pombe by Puromycin

To investigate protein translocation in eukaryotes, we reconstituted a protein translocation system using the permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe. The precursor of a sex pheromone of Saccharomyces cerevisiae, prepro-α-factor, was translocated across the endoplasmic reticulum (ER) of S. pombe posttranslationally, and glycosylated to the same extent as in the ER of S. cerevisiae. This suggested that the size of N-linked core-oligosaccharide in the ER of S. pombe is similar to that in S. cerevisiae. This translocation into the ER of S. pombe was inhibited by puromycin, but the translocation in the P-cells of S. cerevisiae was not inhibited. This difference in sensitivity to puromycin was due to the membrane but not the cytosolic fraction. Our results suggested that the translocation machinery of S. pombe was sensitive to puromycin and different from that of S. cerevisiae.     

Nonsense suppression in Schizosaccharomyces pombe: the S. pombe Sup3-e tRNASerUGA gene is active in S. cerevisiae

Summary The gene encoding the efficient UGA suppressor sup3-e of Schizosaccharomyces pombe was isolated by in vivo transformation of Saccharomyces cerevisiae UGA mutants with S. pombe sup3-e DNA. DNA from a clone bank of EcoRI fragments from a S. pombe sup3-e strain in the hybrid yeast vector YRp17 was used to transform the S. cerevisiae multiple auxotroph his4-260 leu2-2 trp1-1 to prototrophy. Transformants were isolated at a low frequency; they lost the ability to grow in minimal medium after passaging in non-selective media. This suggested the presence of the suppressor gene on the non-integrative plasmid. Plasmid DNA, isolated from the transformed S. cerevisiae cells and subsequently amplified in E. coli, transformed S. cerevisiae his4-260 leu2-2 trp1-1 to prototrophy. In this way a 2.4 kb S. pombe DNA fragment carrying the sup3-e gene was isolated. Sequence analysis revealed the presence of two tRNA coding regions separated by a spacer of only seven nucleotides. The sup3-e tRNA _Ser ^UGA tRNA gene is followed by a sequence coding for the initiator tRNA^Met. The transformation results demonstrate that the cloned S. pombe UGA suppressor is active in S. cerevisiae UGA mutant strains.     

Funcelase — An efficient preparation for the isolation of reversion competent protoplasts from yeasts

Summary The lytic preparation Funcelase was shown to be capable of releasing protoplasts from exponential phase cells ofCandida albicans, Kluyveromyces lactis, Saccharomyces cerevisiae, Saccharomycopsis fibuligera andSchizosaccharomyces pombe. The protoplasts so produced displayed reversion frequencies far superior to those isolated by treatment with Novozym 234 or Suc d'Helix pomatia.     

Callus regeneration from Trifolium subterraneum protoplasts and enhanced protoplast division by low-voltage treatment and nurse cells

Seedling and suspension culture protoplasts of subterranean clover (Trifolium subterraneum L.) were successfully cultured in semi-solid drops of calcium alginate and ultrafiltered liquid medium. Protoplast-derived subterranean-clover colonies developed as the osmolality was lowered over three steps. Callus was established from these colonies. Calli derived from protoplasts have failed to regenerate on a range of media. The frequency of dividing subterranean-clover protoplasts was increased in the presence of lucerne (Medicago sativa L.) nurse cells. Low-voltage treatments (200 mV) for the first 16–132 hours of culture also resulted in a 100% increase in the frequency of dividing protoplasts.