Improved gene-targeting efficiency upon starvation in Saccharomycopsis.

Commonly used fungal transformation protocols rely on the use of either electroporation or the lithium acetate/single strand carrier DNA/Polyethylene glycol/heat shock method. We have used the latter method previously in establishing DNA-mediated transformation in Saccharomycopsis schoenii, a CTG-clade yeast that exhibits necrotrophic mycoparasitism. To elucidate the molecular mechanisms of predation by Saccharomycopsis we aim at gene-function analyses to identify virulence-related pathways and genes. However, in spite of a satisfactory transformation efficiency our efforts were crippled by high frequency of ectopic integration of disruption cassettes. Here, we show that overnight starvation of S. schoenii cells, while reducing the number of transformants, resulted in a substantial increase in gene-targeting via homologous recombination. To demonstrate this, we have deleted the S. schoenii CHS1, HIS3 and LEU2 genes and determined the required size of the flanking homology regions. Additionally, we complemented the S. schoenii leu2 mutant with heterologous LEU2 gene from Saccharomycopsis fermentans. To demonstrate the usefulness of our approach we also generated a S. fermentans leu2 strain, suggesting that this approach may have broader applicability.

Determining the changes in metabolites of Dendrobium officinale juice fermented with starter cultures containing Saccharomycopsis fibuligera FBKL2.8DCJS1 and Lactobacillus paracasei FBKL1.3028 through untargeted metabolomics.

BACKGROUND: The present study aimed to investigate the changes in volatile components and metabolites of Dendrobium officinale (D. officinale) juice fermented with starter cultures containing Saccharomycopsis fibuligera and Lactobacillus paracasei at 28 ℃ for 15 days and post-ripened at 4 ℃ for 30 days using untargeted metabolomics of liquid chromatography-mass spectrometry (LC-MS) and headspace solid-phase microextraction-gas chromatography (HS-SPME-GC-MS) before and after fermentation. RESULTS: The results showed that the alcohol contents in the S. fibuligera group before fermentation and after fermentation were 444.806 ± 10.310 µg/mL and 510.999 ± 38.431 µg/mL, respectively. Furthermore, the alcohol content in the fermentation broth group inoculated with the co-culture of L. paracasei + S. fibuligera was 504.758 ± 77.914 µg/mL, containing a significant amount of 3-Methyl-1-butanol, Linalool, Phenylethyl alcohol, and 2-Methyl-1-propanol. Moreover, the Ethyl L (-)-lactate content was higher in the co-culture of L. paracasei + S. fibuligera group (7.718 ± 6.668 µg/mL) than in the L. paracasei (2.798 ± 0.443 µg/mL) and S. fibuligera monoculture groups (0 µg/mL). The co-culture of L. paracasei + S. fibuligera significantly promoted the metabolic production of ethyl L (-)-lactate in D. officinale juice. The differential metabolites screened after fermentation mainly included alcohols, organic acids, amino acids, nucleic acids, and their derivatives. Twenty-three metabolites, including 11 types of acids, were significantly up-regulated in the ten key metabolic pathways of the co-culture group. Furthermore, the metabolic pathways, such as pentose and glucuronate interconversions, the biosynthesis of alkaloids derived from terpenoid and polyketide, and aminobenzoate degradation were significantly up-regulated in the co-culture group. These three metabolic pathways facilitate the synthesis of bioactive substances, such as terpenoids, polyketides, and phenols, and enrich the flavor composition of D. officinale juice. CONCLUSIONS: These results demonstrate that the co-culture of L. paracasei + S. fibuligera can promote the flavor harmonization of fermented products. Therefore, this study provides a theoretical basis for analyzing the flavor of D. officinale juice and the functional investigation of fermentation metabolites.

Genetic diversity and population structure of the amylolytic yeast Saccharomycopsis fibuligera associated with Baijiu fermentation in China.

The amylolytic yeast Saccharomycopsis fibuligera is a predominant species in starters and the early fermentation stage of Chinese liquor (Baijiu). However, the genetic diversity of the species remains largely unknown. Here we sequenced the genomes of 97 S. fibuligera strains from different Chinese Baijiu companies. The genetic diversity and population structure of the strains were analyzed based on 1,133 orthologous genes and the whole genome single nucleotide polymorphisms (SNPs). Four main lineages were recognized. One lineage contains 60 Chinese strains which are exclusively homozygous with relatively small genome sizes (18.55-18.72 Mb) and low sequence diversity. The strains clustered in the other three lineages are heterozygous with larger genomes (21.85-23.72 Mb) and higher sequence diversity. The genomes of the homozygous strains showed nearly 100% coverage with the genome of the reference strain KPH12 and the sub-genome A of the hybrid strain KJJ81 at the above 98% sequence identity level. The genomes of the heterozygous strains showed nearly 80% coverage with both the sub-genome A and the whole genome of KJJ81, suggesting that the Chinese heterozygous strains are also hybrids with nearly 20% genomes from an unidentified source. Eighty-three genes were found to show significant copy number variation between different lineages. However, remarkable lineage specific variations in glucoamylase and α-amylase activities and growth profiles in different carbon sources and under different environmental conditions were not observed, though strains exhibiting relatively high glucoamylase activity were mainly found from the homozygous lineage.

Molecular characterization of the Saccharomycopsis fibuligera ATF genes, encoding alcohol acetyltransferase for volatile acetate ester formation.

Aroma ester components produced by fermenting yeast cells via alcohol acetyltransferase (AATase)-catalyzed intracellular reactions are responsible for the fruity character of fermented alcoholic beverages, such as beer and wine. Acetate esters are reportedly produced at relatively high concentrations by non-Saccharomyces species. Here, we identified 12 ATF orthologues (SfATFs) encoding putative AATases, in the diploid genome of Saccharomycopsis fibuligera KJJ81, an isolate from wheat-based Nuruk in Korea. The identified SfATF proteins (SfAtfp) display low sequence identities with S. cerevisiae Atf1p (between 13.3 and 27.0%). All SfAtfp identified, except SfAtf(A)4p and SfAtf(B)4p, contained the activation domain (HXXXD) conserved in other Atf proteins. Culture supernatant analysis using headspace gas chromatography mass spectrometry confirmed that the recombinant S. cerevisiae strains expressing SfAtf(A)2p, SfAtf(B)2p, and SfAtf(B)6p produced high levels of isoamyl and phenethyl acetates. The volatile aroma profiles generated by the SfAtf proteins were distinctive from that of S. cerevisiae Atf1p, implying difference in the substrate preference. Cellular localization analysis using GFP fusion revealed the localization of SfAtf proteins proximal to the lipid particles, consistent with the presence of amphipathic helices at their N- and C-termini. This is the first report that systematically characterizes the S. fibuligera ATF genes encoding functional AATases responsible for acetate ester formation using higher alcohols as substrate, demonstrating their biotechnological potential for volatile ester production.

Arabinoxylo- and Arabino-Oligosaccharides-Specific α-L-Arabinofuranosidase GH51 Isozymes from the Amylolytic Yeast Saccharomycopsis fibuligera.

Two genes encoding probable α-L-arabinofuranosidase (E.C. 3.2.1.55) isozymes (ABFs) with 92.3% amino acid sequence identity, ABF51A and ABF51B, were found from chromosomes 3 and 5 of Saccharomycopsis fibuligera KJJ81, an amylolytic yeast isolated from Korean wheat-based nuruk, respectively. Each open reading frame consists of 1,551 nucleotides and encodes a protein of 517 amino acids with the molecular mass of approximately 59 kDa. These isozymes share approximately 49% amino acid sequence identity with eukaryotic ABFs from filamentous fungi. The corresponding genes were cloned, functionally expressed, and purified from Escherichia coli. SfABF51A and SfABF51B showed the highest activities on p-nitrophenyl arabinofuranoside at 40~45°C and pH 7.0 in sodium phosphate buffer and at 50°C and pH 6.0 in sodium acetate buffer, respectively. These exo-acting enzymes belonging to the glycoside hydrolase (GH) family 51 could hydrolyze arabinoxylo-oligosaccharides (AXOS) and arabino-oligosaccharides (AOS) to produce only L-arabinose, whereas they could hardly degrade any polymeric substrates including arabinans and arabinoxylans. The detailed product analyses revealed that both SfABF51 isozymes can catalyze the versatile hydrolysis of α-(1,2)-and α-(1,3)-L-arabinofuranosidic linkages of AXOS, and α-(1,2)-, α-(1,3)-, and α-(1,5)-linkages of linear and branched AOS. On the contrary, they have much lower activity against the α-(1,2)-and α-(1,3)-double-substituted substrates than the single-substituted ones. These hydrolases could potentially play important roles in the degradation and utilization of hemicellulosic biomass by S. fibuligera.

A script for initiating molecular biology studies with non-conventional yeasts based on Saccharomycopsis schoenii.

Non-conventional yeasts (NCYs), i.e. all yeasts other than Saccharomyces cerevisiae, are emerging as novel production strains and gain more and more attention to exploit their unique properties. Yet, these yeasts can hardly compete against the advanced methodology and genetic tool kit available for exploiting and engineering S. cerevisiae. Currently, for many NCYs one has to start from scratch to initiate molecular genetic manipulations, which is often time consuming and not straight-forward. More so because utilization of S. cerevisiae tools based on short-flank mediated homologous recombination or plasmid biology are not readily applicable in NCYs. Here we present a script with discrete steps that will lead to the development of a basic and expandable molecular toolkit for ascomycetous NCYs and will allow genetic engineering of novel platform strains. For toolkit development the highly efficient in vivo recombination efficiency of S. cerevisiae is utilized in the generation and initial testing of tools. The basic toolkit includes promoters, reporter genes, selectable markers based on dominant antibiotic resistance genes and the generation of long-flanking homology disruption cassettes. The advantage of having pretested molecular tools that function in a heterologous host facilitate NCY strain manipulations. We demonstrate the usefulness of this script on Saccharomycopsis schoenii, a predator yeast with useful properties in fermentation and fungal biocontrol.

Multi-omics characterization of the necrotrophic mycoparasite Saccharomycopsis schoenii.

Pathogenic yeasts and fungi are an increasing global healthcare burden, but discovery of novel antifungal agents is slow. The mycoparasitic yeast Saccharomycopsis schoenii was recently demonstrated to be able to kill the emerging multi-drug resistant yeast pathogen Candida auris. However, the molecular mechanisms involved in the predatory activity of S. schoenii have not been explored. To this end, we de novo sequenced, assembled and annotated a draft genome of S. schoenii. Using proteomics, we confirmed that Saccharomycopsis yeasts have reassigned the CTG codon and translate CTG into serine instead of leucine. Further, we confirmed an absence of all genes from the sulfate assimilation pathway in the genome of S. schoenii, and detected the expansion of several gene families, including aspartic proteases. Using Saccharomyces cerevisiae as a model prey cell, we honed in on the timing and nutritional conditions under which S. schoenii kills prey cells. We found that a general nutrition limitation, not a specific methionine deficiency, triggered predatory activity. Nevertheless, by means of genome-wide transcriptome analysis we observed dramatic responses to methionine deprivation, which were alleviated when S. cerevisiae was available as prey, and therefore postulate that S. schoenii acquired methionine from its prey cells. During predation, both proteomic and transcriptomic analyses revealed that S. schoenii highly upregulated and translated aspartic protease genes, probably used to break down prey cell walls. With these fundamental insights into the predatory behavior of S. schoenii, we open up for further exploitation of this yeast as a biocontrol yeast and/or source for novel antifungal agents.

Bioformation of Volatile and Nonvolatile Metabolites by Saccharomycopsis fibuligera KJJ81 Cultivated under Different Conditions-Carbon Sources and Cultivation Times.

Saccharomycopsis fibuligera KJJ81 isolated from nuruk is an amylolytic yeast that is widely used as a microbial starter in various fermented foods. Volatile and nonvolatile metabolites of S. fibuligera KJJ81 were investigated according to different carbon sources and cultivation times using a nontargeted metabolomic approach. Partial-least-squares discriminant analysis was applied to determine the major metabolites, which were found to be closely related to the clustering and discrimination of S. fibuligera KJJ81 samples. Some volatile metabolites derived from phenylalanine, such as 2-phenylethanol, 2-phenylethyl acetate, and ethyl phenylacetate, were predominantly found in cultivation medium containing glucose (YPD medium). In addition, the level of 2-phenylethanol increased continuously with the cultivation time. In terms of nonvolatile metabolites, carbohydrates (mannose, arabitol, and mannitol), fatty acids (palmitic acid and stearic acid), organic acids (oxalic acid and succinic acid), and amino acids (isoleucine, serine, alanine, glutamic acid, glycine, proline, phenylalanine, and threonine) were the main contributors to S. fibuligera KJJ81 samples cultivated in YPD medium according to cultivation time. These results show that the formation of volatile and nonvolatile metabolites of S. fibuligera KJJ81 can be significantly affected by both the carbon sources and the cultivation time.

Comparison of volatile and non-volatile metabolites in rice wine fermented by Koji inoculated with Saccharomycopsis fibuligera and Aspergillus oryzae.

This study investigated volatile and nonvolatile metabolite profiles of makgeolli (a traditional rice wine in Korea) fermented by koji inoculated with Saccharomycopsis fibuligera and/or Aspergillus oryzae. The enzyme activities in koji were also examined to determine their effects on the formation of metabolites. The contents of all 18 amino acids detected were the highest in makgeolli fermented by S. fibuligera CN2601-09, and increased after combining with A. oryzae CN1102-08, unlike the contents of most fatty acids. On the other hand, major volatile metabolites were fusel alcohols, acetate esters, and ethyl esters. The contents of most fusel alcohols and acetate esters were the highest in makgeolli fermented by S. fibuligera CN2601-09, for which the protease activity was the highest, leading to the largest amounts of amino acods. The makgeolli samples fermented only by koji inoculated with S. fibuligera could be discriminated on PCA plots from the makgeolli samples fermented in combination with A. oryzae. In the case of nonvolatile metabolites, all amino acids and some metabolites such as xylose, 2-methylbenzoic acid, and oxalic acid contributed mainly to the characteristics of makgeolli fermented by koji inoculated with S. fibuligera and A. oryzae. These results showed that the formations of volatile and nonvolatile metabolites in makgeolli can be significantly affected by microbial strains with different enzyme activities in koji. To our knowledge, this study is the first report on the effects of S. fibuligera strains on the formation of volatile and non-volatile metabolites in rice wine, facilitating their use in brewing rice wine.

Characterization of biosurfactant from yeast using residual soybean oil under acidic conditions and their use in metal removal processes.

This study aimed at the production of biosurfactants from yeasts under acidic conditions using residual soybean oil as a carbon source, as well as the biosurfactant produced in the solubilization of metals in sewage sludge. The yeast Meyerozyma guilliermondii was considered the best producer in both pH 4.0 and 2.0; therefore, the product obtained by this yeast was characterized by Fourier transform infrared (FT-IR) and 1H nuclear magnetic resonance (NMR) spectroscopy. Moreover, it was applied in metal removal assays in anaerobic sewage sludge. The spectra obtained in FT-IR suggested that M. guilliermondii's biosurfactant had a similar structure to glycolipids from the sophorolipid class, and it was confirmed by 1H NMR spectroscopy. In the bioleaching assays, the application of biosurfactant (2%) produced by M. guilliermondii with pH adjusted to 2.0 was able to solubilize 15.9% of cadmium from the sewage sludge.

Biosurfactant production by yeasts isolated from hydrocarbon polluted environments.

Thirty-two yeast isolates were retrieved from four soil samples collected from hydrocarbon-polluted locations of Hisar, Haryana, using enrichment culture technique with 1% (v/v) diesel as carbon source. Total nine isolates showing blood agar haemolysis were screened further for biosurfactant production. Yeast isolate, YK32, gave highest 8.4-cm oil displacement which was found to be significantly higher as compared to positive control, 0.2% (w/v) SDS (6.6 cm), followed by 6.2 and 6.0 cm by isolates YK20 and YK21, respectively. Maximum emulsification index was obtained in case of isolates YK20 and YK21 measuring 53.8%, after 6 days of incubation utilizing glucose as carbon source, whereas isolate YK32 was found to be reducing surface tension up to 93 dynes/cm and presented 99.6% degree of hydrophobicity. Olive oil has supported maximum surface tension reduction in isolates YK32 and YK21 equivalent to 53 and 48 dynes/cm and gave 88.3 and 88.5% degree of hydrophobicity, respectively. Diesel was not preferred as carbon source by most of the isolates except YK28 which generated 5.5-cm oil displacement, 25% emulsification index, reduced surface tension to the level of 38 dynes/cm and presented 89% degree of hydrophobicity. Conclusively, isolates YK20, YK21, YK22 and YK32 were marked as promising biosurfactant producers and were subjected to identification. Based on microscopic examination and biochemical peculiarities, isolates YK21 and YK22 might be identified as Candida spp., whereas, isolates YK20 and YK32 might be identified as Saccharomycopsis spp. and Brettanomyces spp., respectively. Interestingly it is the first report indicating Saccharomycopsis spp. and Brettanomyces spp. as a potential biosurfactant producer.

A diversity study of Saccharomycopsis fibuligera in rice wine starter nuruk, reveals the evolutionary process associated with its interspecies hybrid.

The amylolytic yeast Saccharomycopsis fibuligera is the predominant yeast in the starter product, nuruk, which is utilized for rice wine production in South Korea. Latest molecular studies explore a recently developed interspecific hybridization among stains of S. fibuligera with a unique genetic feature. However, the origin of the natural hybridization occurrence is still unclear. Thus, to respectively distinguish parental and hybrid strains, specific primer sets were applied on 141 yeast strains isolated from different nuruk samples fermented in different provinces. Sixty-seven strains were defined accordingly as parental species with genome A while 8 strains were defined as hybrid strains. Unexpectedly, another parental species with genome B could not be found among the strain pools yet. Furthermore, it was observed that hybrid strains are phenotypically different from A genome strains; asci containing tetrad ascospores were observed in A genome strains more frequent than in hybrid strains. Nevertheless, hybrid strains were slightly more thermotolerant than A genome strains. Interestingly, all hybrid strains were located only in Jeju province. Based on these sets of data, we speculated that the unique climate of Jeju province might play an evolutionary role in the interspecific hybridization between A genome strains, as well as the unculturable allopatric B genome strains.

Biocontrol of ochratoxigenic moulds (Aspergillus ochraceus and Penicillium nordicum) by Debaryomyces hansenii and Saccharomycopsis fibuligera during speck production.

Speck is a meat product obtained from the deboned leg of pork that is salted, smoked and seasoned for four to six months. During speck seasoning, Eurotium rubrum and Penicillium solitum grow on the surface and collaborate with other moulds and tissue enzymes to produce the typical aroma. Both of these strains usually predominate over other moulds. However, moulds producing ochratoxins, such as Aspergillus ochraceus and Penicillium nordicum, can also co-grow on speck and produce ochratoxin A (OTA). Consequently, speck could represent a potential health risk for consumers. Because A. ochraceus and P. nordicum could represent a problem for artisanal speck production, the aim of this study was to inhibit these mould strains using Debaryomyces hansenii and Saccharomycopsis fibuligera. Six D. hansenii and six S. fibuligera strains were tested in vitro to inhibit A. ochraceus and P. nordicum. The D. hansenii DIAL 1 and S. fibuligera DIAL 3 strains demonstrated the highest inhibitory activity and were selected for in vivo tests. The strains were co-inoculated on fresh meat cuts for speck production with both of the OTA-producing moulds prior to drying and seasoning. At the end of seasoning (six months), OTA was not detected in the speck treated with both yeast strains. Because the yeasts did not adversely affect the speck odour or flavour, the strains are proposed as starters for the inhibition of ochratoxigenic moulds.

Effects of innovative and conventional sanitizing treatments on the reduction of Saccharomycopsis fibuligera defects on industrial durum wheat bread.

Wickerhamomyces anomalus, Hyphopichia burtonii and Saccharomycopsis fibuligera are spoilage yeasts causing chalk mold defects on sliced bread packaged under modified atmosphere. The first objective of this study, carried out in a bread-making company for two consecutive years, was to genetically identify yeasts isolated from spoiled sliced bread in Modified Atmosphere Packaging (MAP) and to determine the dominant species among identified strains. The second objective was to evaluate the effects of hydrogen peroxide and silver solution 12% (HPS) treatment in the leavening cells and cooling chambers, in comparison with the conventional Ortho-Phenylphenol (OPP) fumigating treatment, on the incidence of chalk defects of the commercialized products. One-hundred percent of the isolated yeasts were identified as S. fibuligera, while H. burtonii and W. anomalus were not detected. Concerning mean water activity (aw) and moisture content values, packaged bread samples were, respectively, included in the range 0.922-0.940 and 33.40-35.39%. S. fibuligera was able to grow in a wide range of temperature (11.5 to 28.5°C) and relative humidity (70.00 to 80.17%) in the processing environments, and product aw<0.94. Compared to OPP, the combined treatment with hydrogen peroxide and silver solution, in association with MAP, reduced to a negligible level yeast contamination of industrial sliced bread. The identification of the spoilage organisms and a comprehensive understanding of the combined effects of aw, pO2/pCO2 inside the packages, environmental conditions and sanitizing treatment on the growth behaviour is essential for future development of adequate preventive process strategies against chalk mold defects.

A strain of Meyerozyma guilliermondii isolated from sugarcane juice is able to grow and ferment pentoses in synthetic and bagasse hydrolysate media.

The search for new microbial strains that are able to withstand inhibitors released from hemicellulosic hydrolysis and are also still able to convert sugars in ethanol/xylitol is highly desirable. A yeast strain isolated from sugarcane juice and identified as Meyerozyma guilliermondii was evaluated for the ability to grow and ferment pentoses in synthetic media and in sugarcane bagasse hydrolysate. The yeast grew in xylose, arabinose and glucose at the same rate at an initial medium pH of 5.5. At pH 4.5, the yeast grew more slowly in arabinose. There was no sugar exhaustion within 60 h. At higher xylose concentrations with a higher initial cell concentration, sugar was exhausted within 96 h at pH 4.5. An increase of 350 % in biomass was obtained in detoxified hydrolysates, whereas supplementation with 3 g/L yeast extract increased biomass production by approximately 40 %. Ethanol and xylitol were produced more significantly in supplemented hydrolysates regardless of detoxification. Xylose consumption was enhanced in supplemented hydrolysates and arabinose was consumed only when xylose and glucose were no longer available. Supplementation had a greater impact on ethanol yield and productivity than detoxification; however, the product yields obtained in the present study are still much lower when compared to other yeast species in bagasse hydrolysate. By the other hand, the fermentation of both xylose and arabinose and capability of withstanding inhibitors are important characteristics of the strain assayed.

Phenolic profile and antioxidant activity of extracts prepared from fermented heat-stabilized defatted rice bran.

Heat-stabilized, defatted rice bran (HDRB) serves as a potential source of phenolic compounds which have numerous purported health benefits. An estimated 70% of phenolics present in rice bran are esterified to the arabinoxylan residues of the cell walls. Release of such compounds could provide a value-added application for HDRB. The objective of this study was to extract and quantify phenolics from HDRB using fermentation technology. Out of 8 organisms selected for rice bran fermentation, Bacillus subtilis subspecies subtilis had the maximum phenolic release of 26.8 mg ferulic acid equivalents (FAE) per gram HDRB. Response surface methodology was used to further optimize the release of rice bran phenolics. An optimum of 28.6 mg FAE/g rice bran was predicted at 168 h, 0.01% inoculation level, and 100 mg HDRB/mL. Fermentation of HDRB for 96 h with B. subtilis subspecies subtilis resulted in a significant increase in phenolic yield, phenolic concentration, and radical scavenging capacity. Fermented rice bran had 4.86 mg gentistic acid, 1.38 mg caffeic acid, 6.03 mg syringic acid, 19.02 mg (-)-epicatechin, 4.08 mg p-courmaric acid, 4.64 mg ferulic acid, 10.04 mg sinapic acid, and 17.59 mg benzoic acid per 100 g fermented extract compared to 0.65 mg p-courmaric acid and 0.36 mg ferulic acid per 100 g nonfermented extract. The high phenolic content and antioxidant activity of fermented HDRB extract indicates that rice bran fermentation under optimized condition is a potential means of meeting the demand for an effective and affordable antioxidant.

Mig1 is involved in mycelial formation and expression of the genes encoding extracellular enzymes in Saccharomycopsis fibuligera A11.

The MIG1 gene of Saccharomycopsis fibuligera A11 was cloned from its genomic DNA using the degenerated primers and inverse PCR. The MIG1 gene (1152bp, accession number: HM450676) encoded a 384-amino acid protein very similar to Mig1s from other fungi. Besides their highly conserved zinc fingers, the Mig1 proteins displayed short conserved motifs of possible significance in glucose repression. The MIG1 gene in S. fibuligera A11 was disrupted by integrating the HPT (hygromycin B phosphotransferase) gene into ORF (Open Reading Frame) of the MIG1 gene. The disruptant A11-c obtained could grow in the media containing hygromycin and 2-deoxy-d-glucose, respectively. α-Amylase, glucoamylse, acid protease and ß-glucosidase production by the disruptant and expression of their genes in the disruptant were greatly enhanced. This confirms that Mig1, the transcriptional repressor, indeed regulates expression of the genes and production of the extracellular enzymes in S. fibuligera A11. At the same time, it was found that cell budding was enhanced and mycelial formation was reduced in the disruptant.

Trehalose accumulation from cassava starch and release by a highly thermosensitive and permeable mutant of Saccharomycopsis fibuligera.

Highly thermosensitive and permeable mutants are the mutants from which intracellular contents can be released when they are incubated both in low osmolarity water and at non-permissive temperature (usually 37°C). After mutagenesis by using nitrosoguanidine, a highly thermosensitive and permeable mutant named A11-b was obtained from Saccharomycopsis fibuligera A11-12, a trehalose overproducer in which the acid protease gene has been disrupted. Of the total trehalose, 73.8% was released from the mutant cells suspended in distilled water after they had been treated at 37°C overnight. However, only 10.0% of the total trehalose was released from the cells of S. fibuligera A11-12 treated under the same conditions. The cell volume of the mutant cells suspended in distilled water and treated at 37°C overnight was much bigger than that of S. fibuligera A11-12 treated under the same conditions. The cell growth and trehalose accumulation of the mutant were almost the same as those of S. fibuligera A11-12 during the cultivation at the flask level and in a 5-l fermentor. Both could accumulate around 28.0% (w/w) trehalose from cassava starch. After purification, the trehalose crystal from the aqueous extract of the mutant was obtained.

Construction of amylolytic industrial brewing yeast strain with high glutathione content for manufacturing beer with improved anti-staling capability and flavor.

Glutathione in beer works as the main antioxidant compounds which correlates with beer flavor stability. High residual sugars in beer contribute to major non-volatile components which correlate to high caloric content. In this work, Saccharomyces cerevisiae GSH1 gene encoding glutamylcysteine synthetase and Scharomycopsis fibuligera ALP1 gene encoding alpha-amylase were co-expressed in industrial brewing yeast strain Y31 targeting at alpha-acetolactate synthase (AHAS) gene (ILV2) and alcohol dehydrogenase gene (ADH2), and new recombinant strain TY3 was constructed. The glutathione content from the fermentation broth of TY3 increased to 43.83 mg/l compared to 33.34 mg/l from Y31. The recombinant strain showed high alpha-amylase activity and utilized more than 46% of starch after 5 days growing on starch as sole carbon source. European Brewery Convention tube fermentation tests comparing the fermentation broth of TY3 and Y31 showed that the flavor stability index increased to 1.3 fold and residual sugar concentration were reduced by 76.8%, respectively. Due to the interruption of ILV2 gene and ADH2 gene, the amounts of off-flavor compounds diacetyl and acetaldehyde were reduced by 56.93% and 31.25%, comparing with the amounts of these from Y31 fermentation broth. In addition, as no drug-resistance genes were introduced to new recombinant strain, consequently, it should be more suitable for use in beer industry because of its better flavor stability and other beneficial characteristics.

Trehalose accumulation from corn starch by Saccharomycopsis fibuligera A11 during 2-l fermentation and trehalose purification.

In this study, corn starch was used as the substrate for cell growth and trehalose accumulation by Saccharomycopsis fibuligera A11. Effect of different aeration rates, agitation speeds, and concentrations of corn starch on direct conversion of corn starch to trehalose by S. fibuligera A11 were examined using a Biostat B2 2-l fermentor. We found that the optimal conditions for direct conversion of corn starch to trehalose by this yeast strain were that agitation speed was 200 rpm, aeration rate was 4.0 l/min, concentration of corn starch was 2.0% (w/v), initial pH was 5.5, fermentation temperature was 30 degrees C. Under these conditions, over 22.9 g of trehalose per 100 g of cell dry weight was accumulated in the yeast cells, cell mass was 15.2 g/l of the fermentation medium, 0.12% (w/v) of reducing sugar, and 0.21% (w/v) of total sugar were left in the fermented medium within 48 h of the fermentation. It was found that trehalose in the yeast cells could be efficiently extracted by the hot distilled water (80 degrees C). After isolation and purification, the crystal trehalose was obtained from the extract of the cells.