FUM1 Gene Research Articles

Differential effect of environmental conditions on the growth and regulation of the fumonisin biosynthetic gene FUM1 in the maize pathogens and fumonisin producers Fusarium verticillioides and Fusarium proliferatum

The effects of ecophysiological factors, temperature and solute potential, on both the growth and the regulation of the fumonisin biosynthetic FUM1 gene were studied and compared in one isolate each of the two closely related fumonisin-producing and maize pathogens Fusarium verticillioides and Fusarium proliferatum. The effect of solute potential and temperature was examined on in vitro mycelia growth and on the expression of the FUM1 gene, quantified by species-specific real-time reverse transcriptase-PCR assays. Although both isolates showed similar two-dimensional profiles of growth, for F. verticillioides, optimal growth conditions were maintained at higher temperatures and lower solute potential values. FUM1 gene expression was markedly induced at 20 degrees C in both isolates, under suboptimal conditions for growth; however, their expression patterns differed in relation to solute potential. Whereas FUM1 expression was induced in response to increasing water stress in the isolate of F. verticillioides, the F. proliferatum one showed a stable expression pattern regardless of water potential conditions. These results suggest a differential regulation of fumonisin biosynthesis in these isolates of the two species that might be related to their different host range, and play an ecological role. Additionally, environmental conditions leading to water stress (drought) might result in increased risk of fumonisin contamination of maize caused by F. verticillioides.

Fumonisin detection and analysis of potential fumonisin‐producing Fusarium spp. in asparagus (Asparagus officinalis L.) in Zhejiang Province of China

Fumonisins are mycotoxins produced by a number of Fusarium species, including several pathogens of asparagus plants. China is one of the largest asparagus producers in the world. In this study, we analysed the contamination of fumonisins and fumonisin-producing fungi in asparagus spear samples from Zhejiang Province, the major asparagus production province in China. The asparagus did not contain a detectable level of fumonisins. However, the recovery of Fusarium in asparagus was 72.7%, including F. proliferatum (40.9%), F. oxysporum (22.7%), F. acuminatum (4.55%) and F. equesti (4.55%). A multiplex PCR targeting the internal transcribed spacer sequence (ITS), translation elongation factor 1-alpha (TEF), and key biosynthetic genes FUM1 and FUM8, was used to simultaneously determine the identity and the biosynthetic ability of the fungal isolates. Fungal isolates containing the FUM genes also produced fumonisins in cultures, ranging from 28 to 4204 microg g(-1). F. proliferatum was the only fumonisin-producing Fusarium species in asparagus. Although no fumonisin contamination was detected in asparagus in the current survey, we found that the majority of samples contained Fusarium spp. Because F. proliferatum is a high fumonisin-producing species, potential health risks for human consumption of asparagus exist, if the appropriate environmental conditions are present for this fungus.

A molecular based strategy for rapid diagnosis of toxigenic Fusarium species associated to cereal grains from Argentina

Fusarium species are worldwide causal agents of ear rot in cereals. Their toxigenic potential is a health risk for both humans and animals. In Argentina, most identification of these fungi has been based on morphological and cross-fertility criteria which are time consuming and require considerable expertise in Fusarium taxonomy and physiology. DNA based approaches have been reported as rapid, sensitive and specific alternatives to identify the main fumonisin and trichothecene-producing Fusarium species. In this work, we used PCR assays and the partial sequence of TEF1-α gene (Translation Elongation Factor-1α) to identify the fumonisin and trichothecene-producing species in Fusarium isolates from diverse regions of Argentina. The relative efficiency and reliability of those methods to improve mycotoxin risk prediction in this country were also assessed. Species-specific PCR assays were targeted toward multicopy IGS (Intergenic Spacer of rDNA units) and on the toxin biosynthetic genes FUM1 (fumonisins) and TRI13 and TRI7 genes (trichothecenes). PCR assays based on FUM1 gene and IGS sequences allowed detection and discrimination of the fumonisin producers Fusarium proliferatum and Fusarium verticillioides. Molecular identification of nonfumonisin producers from Gibberella fujikuroi species complex was possible after determination of TEF1-α gene sequences, which indicated the presence of Fusarium subglutinans, Fusarium andiyazi and Fusarium thapsinum. TEF-1α gene sequences also allowed discrimination of the different species of the Fusarium graminearum complex ( F. graminearum sensu lato) as F. graminearum sensu stricto, Fusarium meridionale and Fusarium boothii. The last two species belonged to NIV chemotype and were detected for the first time in the subtropical region of Argentina while F. graminearum sensu stricto was DON producer only, which was also confirmed by specific PCR assays based on TRI137/ TRI7 genes. Our results indicated that the PCR assays evaluated in this work are reliable diagnostic tools to detect the main toxigenic Fusarium species associated to cereal grains in Argentina. An extensive epidemiological survey based on the approach presented in this work is currently in progress to know the mycotoxigenic hazard of Fusarium species in cereal grains from the subtropical region of Argentina.

Genetic variability and Fumonisin production by Fusarium proliferatum

Fusarium proliferatum is together with Fusarium verticillioides the main source of fumonisins, a health risk mycotoxin, contaminating agro-products. Contrary to F. verticillioides, it colonizes a wide range of host plants besides maize, such as wheat or barley among others, in particular in certain regions (Southern Europe). The phylogenetic study performed in this work using a wide sample of isolates from diverse hosts and origins revealed a high variability, while no host preferences could be sustained. A real time RT-PCR assay was also developed specific for F. proliferatum on the basis on fumonisin biosynthetic gene, FUM1, which allowed discrimination from F. verticillioides. FUM1 gene expression showed a high and significant correlation (0.77) with fumonisin production, representing a valuable tool for specific and sensitive diagnosis of metabolically active fumonisin-producing F. proliferatum isolates and for evaluating the influence on environmental conditions on FUM1 gene regulation. The ability to produce fumonisins was also widely distributed indicating that F. proliferatum can represent a risk for health similarly to F. verticillioides. Moreover, the wide range of plants susceptible to colonization by F. proliferatum suggests that the impact of fumonisin risk in a number of commodities might need a revision.

Genetic Variability of Phytopathogenic Fusarium proliferatum Associated with Crown Rot in Asparagus officinalis

Abstract Fusarium proliferatum (teleomorph: Gibberella intermedia) is a causal agent of crown rot of Asparagus officinalis and is one potential fumonisin‐producing species within the genus Fusarium. It colonizes roots and crowns of asparagus plants, but could also be isolated from symptomless asparagus spears. Fusarium proliferatum isolates obtained from perennial asparagus plantings from Austria and Germany were included in a study on detectability and variability of two essential genes of the fumonisin‐gene cluster. Genetic fingerprinting of 45 isolates revealed 14 different fingerprint groups, indicating genetic heterogenicity of F. proliferatum. Most isolates differentiated into three main fingerprint clusters, but no association was found between fingerprint group and origin of the isolates. By gene‐specific PCR it was shown that, in 25 isolates tested, both initial genes of the fumonisin biosynthetic pathway –FUM1, encoding a polyketide synthase and FUM8, a gene for a putative aminoacyl transferase – were detectable. This suggests that these isolates were able to produce fumonisins and could contribute to the detected contamination in originating asparagus spears with this mycotoxin. Thus, early detection of FUM‐genes in F. proliferatum‐colonized asparagus may be suited to prevent uptake of fumonisin contaminated food with the human diet. Restriction fragment length polymorphism analysis (PCR‐RFLP) of the amplified FUM gene fragments revealed little sequence variability, suggesting a conserved structure of these genes within this species. However, sequence analysis confirmed intraspecific nucleotide polymorphisms of these genes.

Fumonisin B1production byFusarium proliferatumstrains isolated fromAllium fistulosumplants and seeds in Japan

To test the fumonisin B(1)-producing ability of Fusarium proliferatum strains isolated from Welsh onion (Allium fistulosum) plants and seeds of commercial cultivars in Japan and to examine the applicability of PCR-based assays to discriminate between fumonisin B(1)-producing and nonproducing isolates. Fumonisin B(1) levels in 20 Fusarium isolates obtained from Welsh onion plants and seeds of seven commercial cultivars were determined by HPLC. Thirteen of the 20 isolates produced fumonisin B(1). PCR assay with FUM1 gene-specific primers amplified a DNA fragment (700 bp) only from fumonisin-producing isolates. Fusarium proliferatum isolates that can produce fumonisin B(1) were often associated with wilted Welsh onion plants and seeds of some commercial cultivars. The PCR assay with FUM1 gene-specific primers has the potential to discriminate between fumonisin B(1)-producing and nonproducing isolates. This study revealed that F. proliferatum producing fumonisin B(1) is associated with Welsh onion plants and that commercial cultivar seeds may be contaminated with the fungus. PCR amplification of FUM1 gene can be a useful tool for the rapid identification of fumonisin B(1)-producing F. proliferatum isolates.

N-starvation stress induced FUM gene expression and fumonisin production is mediated via the HOG-type MAPK pathway in Fusarium proliferatum

During cultivation of a wild type strain of Fusarium proliferatum on ammonium dihydrogen phosphate containing defined medium, expression levels of FUM1 and FUM8, members of the fumonisin biosynthesis gene cluster significantly increased when ammonium ion concentration of the culture medium decreased below 10 mM, indicating that N-depletion triggers the fumonisin biosynthesis genes. Deletion of Fphog1, a HOG-type MAP kinase gene resulted in further increases in FUM1 and FUM8 expression under nitrogen starvation (absence of any N-source) conditions. Fumonisin B1 (FB1) production paralleled with increased FUM gene expression: significant amounts of FB1 were measured in culture filtrates of the ΔFphog1 deleted mutant after five days culturing, whereas only traces of FB1 could be detected in filtrates of the wild type and the restored strain (R1) complemented with the wild-type Fphog1–24 gene. N-starvation strongly retarded the growth of the ΔFphog1 mutant in comparison to wild type. The up-regulation of fumonisin biosynthesis genes in the ΔFphog1 mutant could be explained by the increased sensitivity of these strains to N-starvation stress that appears in the absence of an intact HOG-type MAPK pathway.

A fumonisin biosynthetic gene cluster in Fusarium oxysporum strain O-1890 and the genetic basis for B versus C fumonisin production

Most species of Fusarium that produce fumonisin mycotoxins produce predominantly B fumonisins (FBs). However, Fusarium oxysporum strain O-1890 produces predominantly C fumonisins (FCs). In this study, the nucleotide sequence of the fumonisin biosynthetic gene ( FUM) cluster in strain O-1890 was determined. The order and orientation of FUM genes were the same as in the previously described clusters in Fusarium verticillioides and Fusarium proliferatum. Coding regions of F. oxysporum and F. verticillioides FUM genes were 88–92% identical, but regions flanking the clusters did not share significant identity. The FUM cluster gene FUM8 encodes an α-oxoamine synthase, and fum8 mutants of F. verticillioides do not produce fumonisins. Complementation of a fum8 mutant with the F. verticillioides FUM8 restored FB production. Complementation with F. oxysporum FUM8 also restored production, but the fumonisins produced were predominantly FCs. These data indicate that different orthologues of FUM8 determine whether Fusarium produces predominantly FBs or FCs.

Functional characterization of Fusarium verticillioides CPP1, a gene encoding a putative protein phosphatase 2A catalytic subunit

Fusarium verticillioides produces the mycotoxin fumonisin B(1) (FB(1)) on maize kernels. In this study, we identified a putative protein phosphatase gene CPP1 in F. verticillioides, and investigated its role in FB(1) regulation. Previous work has shown that CPP1 expression is elevated in an FB(1)-suppressing genetic background. Thus, we hypothesized that CPP1 is negatively associated with FB(1) production. To test this hypothesis, we generated a CPP1 knockout mutant, PP179, and studied the effects of gene deletion on FB(1) biosynthesis and fungal development. PP179 showed elevated expression of FUM genes, and in turn produced higher levels of FB(1) than the wild-type progenitor. Other significant mutant phenotypes included reduced radial growth on agar plates, reduced conidia germination rates, significantly increased macroconidia formation, and hyphal swelling. To verify that these phenotypes were directly due to CPP1 deletion, we complemented PP179 with the wild-type CPP1 gene. The complemented strain PPC4 showed FUM1 expression and FB(1) production similar to that of the wild-type, providing evidence that CPP1 is negatively associated with FB(1) biosynthesis. Other PP179 phenotypes, such as macroconidiation and hyphal swelling, were also restored to that of wild-type progenitor. Furthermore, we complemented F. verticillioides PP179 strain with Neurospora crassa wild-type ppe-1 gene, demonstrating that Cpp1 and PPE-1 proteins are functionally conserved. Pleiotropic effects of CPP1 deletion led us to hypothesize that CPP1 is associated with multiple downstream signalling pathways in F. verticillioides. Identification and functional characterization of downstream Cpp1-interacting proteins are necessary to better understand the complex regulatory mechanisms associated with Cpp1.

Sequence variability in the FUM1 gene of Fusarium verticillioides strains

Fumonisins are mycotoxins, produced mainly by Fusarium verticillioides, that are potentially carcinogenic to humans and toxic to animals. Synthesis of these toxins is directed by a cluster of 15 genes, among which FUM1 is the largest; it encodes a polyketide synthase. This enzyme probably catalyzes the synthesis of a polyketide that forms a large portion of the fumonisin structure. In this study, 27 strains possessing the FUM1 gene, as determined by polymerase chain reaction, were analyzed. A portion of the FUM1 gene was amplified and sequenced from 6 of 27 Brazilian strains isolated from corn and sorghum. The sequence similarity for the six F. verticillioides strains was almost 100%.

The putative monomeric G‐protein GBP1 is negatively associated with fumonisin B1 production in Fusarium verticillioides

SUMMARY Fumonisin B(1) (FB(1)) is a mycotoxin produced by Fusarium verticillioides that contaminates maize. FB(1) has been linked to a number of human and animal mycotoxicoses worldwide. Despite its significance, our understanding of the FB(1) biosynthesis regulatory mechanisms is limited. Here, we describe F. verticillioides GBP1, encoding a monomeric G-protein, and its role in FB(1) biosynthesis. GBP1 was discovered as an expressed sequence tag (EST) up-regulated in the F. verticillioides fcc1 mutant that showed reduced conidiation and no FB(1) biosynthesis when grown on maize kernels. Sequence analysis showed that GBP1 encodes a putative 368-amino-acid protein with similarity to DRG and Obg subclasses of G-proteins that are involved in development and stress responses. A GBP1 knockout mutant (Deltagbp1) exhibited normal growth, but increased FB(1) production (> 58%) compared with the wild-type when grown on corn kernels. Complementation of Deltagbp1 with the wild-type GBP1 gene restored FB(1) production levels to that of the wild-type. Our data indicate that GBP1 is negatively associated with FB(1) biosynthesis but not with conidiation in F. verticillioides. The deletion of GBP1 led to up-regulation of key FB(1) biosynthetic genes, FUM1 and FUM8, suggesting that the increased FB(1) production in Deltagbp1 is due to over-expression of FUM genes.

Multiplex PCR‐based detection of potential fumonisin‐producing Fusarium in traditional African vegetables

Culture-independent methods employed in fungal genetic studies using in vitro amplification (PCR) and analysis of specific genes or gene fragments have proved to be useful for detection, identification, and molecular taxonomy of various plant pathogens including Fusarium spp. This approach may be faster than culture-dependent methods, and could especially be of value for the rapid detection of slow-growing toxin-producing species in food samples. The present study was aimed at the development and evaluation of multiplex PCR-based methods for the detection and identification of potential fumonisin-producing Fusarium spp. in traditional morogo-leafy vegetables supplementing the maize-based staple diet of rural communities in South Africa. In these rural subsistence settings, some morogo plants grow as weeds in maize fields where they might become contaminated with potential fumonisin-producing Fusarium strains before being collected for consumption. Substances released by senescent vegetables could induce toxin production during storage. Using fumonisin-positive MRC Fusarium verticillioides strains as reference cultures, the following primer sets for the detection of specific gene fragments in fumonisin-positive Fusarium spp. were evaluated: (i) the conserved transcription elongation factor gene (EF-1), (ii) the FUM1 gene encoding polyketide synthase for fumonisin B1 production, and (iii) 18S rRNA gene. Preliminary results indicated that, these DNA fragments were amplified from MRC reference strains as well as Fusarium spp. isolated from morogo. The annealing temperature for the multiplex PCR was 55 degrees C and each reaction contained 25 pmol of each of the primer sets EF and FUM1 and 12.5 pmol of the 18S primer set. The detection limit of the individual primers was up to 1 ng and for the multiplex up to 10 ng. This demonstrates the potential of this method for the detection of potential fumonisin-positive strains.

Fumonisin Production by Fusarium verticillioides Strains Isolated from Maize in Mexico and Development of a Polymerase Chain Reaction to Detect Potential Toxigenic Strains in Grains

Fumonisins are mycotoxins produced by Fusarium verticillioides (Sacc. Nirenberg) in maize (Zea mays L.), a staple crop in Mexico. In this study, we report the isolation and identification of 67 Fusarium strains isolated from maize kernels collected in Northwest and Central Mexico. The strains were characterized regarding fumonisin B(1) production and the presence of the FUM1 gene. F. verticillioides was the predominant species isolated in both geographic regions, but the isolates from Northwest Mexico produced higher levels of fumonisin. A polymerase chain reaction (PCR)-based method, to detect a region of the FUM1 gene involved in fumonisin biosynthesis, was developed and employed to detect mycotoxigenic fungi in pure culture and in contaminated maize. The presence of the FUM1 gene was associated with fumonisin production in most isolates, except seven that did not synthesize fumonisin but contained the gene in their genome. The PCR method allowed the direct detection of fungal contamination in ground corn and could be employed to screen for the presence of potential mycotoxigenic fusaria.

Comparative analysis of 87,000 expressed sequence tags from the fumonisin-producing fungus Fusarium verticillioides

Fusarium verticillioides (teleomorph Gibberella moniliformis) is a pathogen of maize worldwide and produces fumonisins, a family of mycotoxins that have been associated with several animal diseases as well as cancer in humans. In this study, we sought to identify fungal genes that affect fumonisin production and/or the plant–fungal interaction. We generated over 87,000 expressed sequence tags from nine different cDNA libraries that correspond to 11,119 unique sequences and are estimated to represent 80% of the genomic complement of genes. A comparative analysis of the libraries showed that all 15 genes in the fumonisin gene cluster were differentially expressed. In addition, nine candidate fumonisin regulatory genes and a number of genes that may play a role in plant–fungal interaction were identified. Analysis of over 700 FUM gene transcripts from five different libraries provided evidence for transcripts with unspliced introns and spliced introns with alternative 3′ splice sites. The abundance of the alternative splice forms and the frequency with which they were found for genes involved in the biosynthesis of a single family of metabolites as well as their differential expression suggest they may have a biological function. Finally, analysis of an EST that aligns to genomic sequence between FUM12 and FUM13 provided evidence for a previously unidentified gene ( FUM20) in the FUM gene cluster.

Discontinuous distribution of fumonisin biosynthetic genes in the Gibberella fujikuroi species complex

Production of the carcinogenic mycotoxins fumonisins has been reported in several Fusarium species, most of which are members of the Gibberella fujikuroi (Gf) complex. In this study, we examined 15 Fusarium species in the Gf complex and 12 other species for fumonisin production and the presence of fumonisin biosynthetic genes (FUM). Among the species within the Gf complex, fumonisin production was detected only in F. fujikuroi, F. globosum, F. proliferatum, F. nygamai, F. oxysporum and F. verticillioides. These five species include members of two of the three major clades delineated in the Gf complex. The FUM genes were detected in these same five species and in F. anthophilum, a member of the third clade. Among the species outside the Gf complex, fumonisin production and FUM genes were detected only in F. oxysporum. Phylogenetic analyses of nucleotide sequences from two FUM gene fragments inferred relationships similar but not identical to those inferred from previous analyses of other genes. The results indicate the FUM genes are discontinuously distributed in the Gf complex and that this distribution gives rise to the differences in the abilities of closely related Fusarium species to produce fumonisins.

FUM13 encodes a short chain dehydrogenase/reductase required for C-3 carbonyl reduction during fumonisin biosynthesis in Gibberella moniliformis.

Fumonisins are polyketide-derived mycotoxins produced by the filamentous fungus Gibberella moniliformis (anamorph Fusarium verticillioides). Wild-type strains of the fungus produce predominantly four B-series fumonisins, designated FB(1), FB(2), FB(3), and FB(4). Recently, a cluster of 15 putative fumonisin biosynthetic genes (FUM) was described in G. moniliformis. We have now conducted a functional analysis of FUM13, a gene in the cluster that is predicted by amino acid sequence similarity to encode a short chain dehydrogenase/reductase (SDR). Mass spectrometric analysis of metabolites from FUM13 deletion mutants revealed that they produce approximately 10% of wild-type levels of B-series fumonisins as well as two previously uncharacterized compounds. NMR analysis revealed that the new compounds are similar in structure to FB(3) and FB(4) but that they have a carbonyl function rather than a hydroxyl function at carbon atom 3 (C-3). These results indicate that the FUM13 protein catalyzes the reduction of the C-3 carbonyl to a hydroxyl group and are the first biochemical evidence directly linking a FUM gene to a specific reaction during fumonisin biosynthesis. The production of low levels of FB(1), FB(2), FB(3), and FB(4), which have a C-3 hydroxyl, by the FUM13 mutants suggests that G. moniliformis has an additional C-3 carbonyl reductase activity but that this enzyme functions less efficiently than the FUM13 protein.

Co-expression of 15 contiguous genes delineates a fumonisin biosynthetic gene cluster in Gibberella moniliformis

Fumonisins are mycotoxins produced by the maize pathogen Gibberella moniliformis and are associated with cancer in rodents. In this study, we determined the nucleotide sequence of a 75-kb region of G. moniliformis DNA and identified 18 heretofore undescribed genes flanking a cluster of five previously identified fumonisin biosynthetic ( FUM) genes. Ten of the newly identified genes downstream of the cluster were coregulated with FUM genes and exhibited patterns of expression that were correlated with fumonisin production. BLASTX analyses indicated that the predicted functions of proteins encoded by the 10 genes were consistent with activities expected for fumonisin biosynthesis or self-protection. These data indicate that the 10 newly identified genes and the previously identified FUM genes constitute a fumonisin biosynthetic gene cluster. Disruption of two of the new genes, encoding longevity assurance factors, had no apparent effect on fumonisin production, but disruption of a third, encoding an ABC transporter, had a subtle effect on ratios of fumonisins produced.

Mitochondrial and Cytosolic Isoforms of Yeast Fumarase Are Derivatives of a Single Translation Product and Have Identical Amino Termini

We have previously proposed that a single translation product of the FUM1 gene encoding fumarase is distributed between the cytosol and mitochondria of Saccharomyces cerevisiae and that all fumarase translation products are targeted and processed in mitochondria before distribution. Alternative models for fumarase distribution have been proposed that require more than one translation product. In the current work (i) we show by using sequential Edman degradation and mass spectrometry that fumarase cytosolic and mitochondrial isoenzymes have an identical amino terminus that is formed by cleavage by the mitochondrial processing peptidase, (ii) we have generated fumarase mutants in which the second potential translation initiation codon (Met-24) has been substituted, yet the protein is processed efficiently and retains its ability to be distributed between the cytosol and mitochondria, and (iii) we show that although a signal peptide is required for fumarase targeting to mitochondria the specific fumarase signal peptide and the sequence immediately downstream to the cleavage site are not required for the dual distribution phenomenon. Our results are discussed in light of our model of fumarase targeting and distribution that suggests rapid folding into an import-incompetent state and retrograde movement of the processed protein back to the cytosol through the translocation pore.

Isolation of High-Malate-Producing Sake Yeasts from Low-Maltose-Assimilating Mutants.

High-malate-producing sake yeasts were isolated from low-maltose-assimilating mutants. A sake yeast Kyokai no. 701 (K-701) was mutagenized with ethyl methanesulfonate. After nystatin treatment, low-maltose-assimilating mutants were selected using a replica plating method. The forty-two mutants obtained were fermented in koji extract medium and almost all of them produced more malate than the parental strain. In a small-scale sake brewing test, several of these mutants produced 2.3 to 6.7 times higher concentrations of malate and 1.5 to 2.1 times higher total acidity than the parental strain. However, these mutants exhibited neither resistance to cycloheximide nor sensitivity to dimethyl succinate, which are used as the phenotypes for isolation of high-malate-producing sake yeasts. The expression levels of the MDH and FUM1 genes in one mutant (M20), which produced the highest amount of malate among the mutants obtained, were analyzed by Northern hybridization. The transcriptional level of the FUM1 gene in strain M20 during sake brewing had a similar profile to that in strain K-701. However, the transcriptional level of the MDH2 gene in strain M20 for days 4 and 8 of malate formation during sake brewing was higher than that in strain K-701.

Isolation of high-malate-producing sake yeasts from low-maltose-assimilating mutants

High-malate-producing sake yeasts were isolated from low-maltose-assimilating mutants. A sake yeast Kyokai no. 701 (K-701) was mutagenized with ethyl methanesulfonate. After nystatin treatment, low-maltose-assimilating mutants were selected using a replica plating method. The forty-two mutants obtained were fermented in koji extract medium and almost all of them produced more malate than the parental strain. In a small-scale sake brewing test, several of these mutants produced 2.3 to 6.7 times higher concentrations of malate and 1.5 to 2.1 times higher total acidity than the parental strain. However, these mutants exhibited neither resistance to cycloheximide nor sensitivity to dimethyl succinate, which are used as the phenotypes for isolation of high-malate-producing sake yeasts. The expression levels of the MDH and FUM1 genes in one mutant (M20), which produced the highest amount of malate among the mutants obtained, were analyzed by Northern hybridization. The transcriptional level of the FUM1 gene in strain M20 during sake brewing had a similar profile to that in strain K-701. However, the transcriptional level of the MDH2 gene in strain M20 for days 4 and 8 of malate formation during sake brewing was higher than that in strain K-701.