Observation of different Li intercalation states and local doping in epitaxial mono- and bilayer graphene on SiC(0001).

Li intercalation is commonly used to enhance the carrier density in epitaxial graphene and mitigate coupling to the substrate. So far, the understanding of the intercalation process, particularly how Li penetrates different layers above the substrate, and its impact on electron transport remains incomplete. Here, we report different phases of Li intercalation and their kinetic processes in epitaxial mono- and bilayer graphene grown on SiC. The distinct doping effects of each intercalation phase are characterized using scanning tunneling spectroscopy. Furthermore, changes in the local conduction regimes are directly mapped by scanning tunneling potentiometry and attributed to different charge transfer states of the intercalated Li. The stable intercalation marked by the formation of Li-Si bonds leads to a significant 56% reduction in sheet resistance of the resulting quasi-free bilayer graphene, as compared to the pristine monolayer graphene.

Integrated genomics and phenotype microarray analysis of Saccharomyces cerevisiae industrial strains for rice wine fermentation and recombinant protein production.

The industrial potential of Saccharomyces cerevisiae has extended beyond its traditional use in fermentation to various applications, including recombinant protein production. Herein, comparative genomics was performed with three industrial S. cerevisiae strains and revealed a heterozygous diploid genome for the 98-5 and KSD-YC strains (exploited for rice wine fermentation) and a haploid genome for strain Y2805 (used for recombinant protein production). Phylogenomic analysis indicated that Y2805 was closely associated with the reference strain S288C, whereas KSD-YC and 98-5 were grouped with Asian and European wine strains, respectively. Particularly, a single nucleotide polymorphism (SNP) in FDC1, involved in the biosynthesis of 4-vinylguaiacol (4-VG, a phenolic compound with a clove-like aroma), was found in KSD-YC, consistent with its lack of 4-VG production. Phenotype microarray (PM) analysis showed that KSD-YC and 98-5 displayed broader substrate utilization than S288C and Y2805. The SNPs detected by genome comparison were mapped to the genes responsible for the observed phenotypic differences. In addition, detailed information on the structural organization of Y2805 selection markers was validated by Sanger sequencing. Integrated genomics and PM analysis elucidated the evolutionary history and genetic diversity of industrial S. cerevisiae strains, providing a platform to improve fermentation processes and genetic manipulation.

Differentiation of Geographical Origin of White and Brown Rice Samples Using NMR Spectroscopy Coupled with Machine Learning Techniques.

Rice (Oryza sativa L.) is a widely consumed food source, and its geographical origin has long been a subject of discussion. In our study, we collected 44 and 20 rice samples from different regions of the Republic of Korea and China, respectively, of which 35 and 29 samples were of white and brown rice, respectively. These samples were analyzed using nuclear magnetic resonance (NMR) spectroscopy, followed by analyses with various data normalization and scaling methods. Then, leave-one-out cross-validation (LOOCV) and external validation were employed to evaluate various machine learning algorithms. Total area normalization, with unit variance and Pareto scaling for white and brown rice samples, respectively, was determined as the best pre-processing method in orthogonal partial least squares-discriminant analysis. Among the various tested algorithms, support vector machine (SVM) was the best algorithm for predicting the geographical origin of white and brown rice, with an accuracy of 0.99 and 0.96, respectively. In external validation, the SVM-based prediction model for white and brown rice showed good performance, with an accuracy of 1.0. The results of this study suggest the potential application of machine learning techniques based on NMR data for the differentiation and prediction of diverse geographical origins of white and brown rice.

Enzyme activity of Aspergillus section Nigri strains isolated from the Korean fermentation starter, nuruk.

Aspergillus section Nigri is a fungus used industrially because of its ability to produce enzymes such as cellulolytic, amylolytic and proteolytic enzymes. In this study, we obtained twenty-eight strains of Aspergillus section Nigri from the traditional Korean fermentation starter, nuruk, which is known as a mixed culture of enzymatic filamentous fungi and yeasts. All strains were identified as Aspergillus section Nigri through combined phylogenetic analysis using partial ß-tubulin and calmodulin gene sequences. The cellulase, amylase and protease activities of Korean strains were measured and compared with ten reference strains of Aspergillus niger. Most Korean strains showed higher cellulolytic activity than reference strains, and Aspergillus neoniger KCN5 showed the highest ß-glucosidase activity. Two-thirds of the Korean strains showed similar levels of α- and glucoamylase activity as the reference strains. The protease activity of Aspergillus section Nigri strains was the highest at pH 3.0, and A. niger KSJ2 showed the highest acidic protease activity. By comparing ten reference strains and twenty-eight Korean strains, our results suggested useful Aspergillus section Nigri strains from nuruk with high enzyme activity, such as KCN5 and KSJ2, and their potential for industrial applications as enzyme producers.

pH Changes Have a Profound Effect on Gene Expression, Hydrolytic Enzyme Production, and Dimorphism in Saccharomycopsis fibuligera.

Saccharomycopsis fibuligera is an amylolytic yeast that plays an important role within nuruk (a traditional Korean fermentation starter) used for the production of makgeolli (Korean rice wine), which is characterized by high acidity. However, the effect of pH change (neutral to acidic) on the yeast cell to hyphal transition and carbohydrate-hydrolyzing enzyme activities for S. fibuligera has not been investigated yet. In this study, S. fibuligera strains were cultured under the different pH conditions, and the effect on the enzyme production and gene expression were investigated. An acidic pH induced a hyphal transition from yeast cell of S. fibuligera KPH12 and the hybrid strain KJJ81. In addition, both strains showed a gradual decrease in the ability to degrade starch and cellulose as the pH went down. Furthermore, a transcriptome analysis demonstrated that the pH decline caused global expression changes in genes, which were classified into five clusters. Among the differentially expressed genes (DEGs) under acidic pH, the downregulated genes were involved in protein synthesis, carbon metabolism, and RIM101 and cAMP-PKA signaling transduction pathways for the yeast-hyphal transition. A decrease in pH induced a dimorphic lifestyle switch from yeast cell formation to hyphal growth in S. fibuligera and caused a decrease in carbohydrate hydrolyzing enzyme production, as well as marked changes in the expression of genes related to enzyme production and pH adaptation. This study will help to elucidate the mechanism of adaptation of S. fibuligera to acidification that occur during the fermentation process of makgeolli using nuruk.

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.

Mycobiome analysis for distinguishing the geographical origins of sesame seeds.

Sesame (Sesamum indicum) is one of the most widely cultivated crops in Asia and Africa. The identification of the geographical origins of sesame seeds is important for the detection of fraudulent samples. This study was conducted to build a prediction model and suggest potential biomarkers for distinguishing the geographical origins of sesame seeds using mycobiome (fungal microbiome) analysis coupled with multivariate statistical analysis. Sesame seeds were collected from 25 cities in Korea, six cities in China, and five sites in other countries (Ethiopia, India, Nigeria, and Pakistan). According to the expression of fungal internal transcribed spacer (ITS) sequences in sesame seeds, 21 fungal genera were identified in sesame seeds from various countries. The optimal partial least squares-discriminant analysis model was established by applying two components with unit variance scaling. Based on seven-fold cross validation, the predictive model had 94.4% (Korea vs. China/other countries), 91.7% (China vs. Korea/other countries), and 88.9% (other countries vs. Korea/China) accuracy in determining the geographical origins of sesame seeds. Alternaria, Aspergillus, and Macrophomina were suggested as the potential fungal genera to differentiate the geographical origins of sesame seeds. This study demonstrated that mycobiome analysis could be used as a complementary method for distinguishing the geographical origins of raw sesame seeds.

Integrated genomic and transcriptomic analysis reveals unique mechanisms for high osmotolerance and halotolerance in Hyphopichia yeast.

The yeast species Hyphopichia is common in nature and strongly competitive under harsh environmental conditions. Here, we characterized Hyphopichia burtonii KJJ43 and H. pseudoburtonii KJS14, which exhibit strong halotolerance, using genomic and transcriptomic analyses. The genomes of H. burtonii and H. pseudoburtonii comprised eight chromosomes with 85.17% nucleotide identity and significant divergence in synteny. Notably, both Hyphopichia genomes possessed extended gene families of amino acid permeases and ATP-binding cassette (ABC) transporters, whose dynamic expression patterns during osmotic stress were revealed using transcriptome profiling. Intriguingly, we found unique features of the HOG pathway activated by Hog1p even under non-osmotic stress conditions and the upregulation of cytosolic Gpd1 protein during osmotic stress. Associated with hyperfilamentation growth under high osmotic conditions, a set of genes in the FLO family with induced expression in response to NaCl, KCl, and sorbitol supplementation were identified. Moreover, comparative transcriptome analysis reveals the NaCl-specific induction of genes involved in amino acid biosynthesis and metabolism, particularly BAT2. This suggests the potential association between oxoacid reaction involving branched-chain amino acids and osmotolerance. The combined omics analysis of two Hyphopichia species provides insights into the novel mechanisms involved in salt and osmo-stress tolerance exploited by diverse eukaryotic organisms.

Unique Metabolic Profiles of Korean Rice According to Polishing Degree, Variety, and Geo-Environmental Factors.

The precise determination of the chemical composition in crops is important to identify their nutritional and functional value. The current study performed a systematic delineation of the rice metabolome, an important staple in Asia, to investigate the following: (1) comparative features between brown and white rice; (2) variety-specific composition (Ilpum vs. Odae); and (3) cultivation of region-dependent metabolic content. Global metabolic profiling and data-driven statistics identified the exclusive enrichment of compounds in brown rice compared to white rice. Next, the authors investigated a variety-governed metabolic phenotype among various geo-environmental factors. Odae, the early-ripening cultivar, showed higher contents of most chemicals compared to the late-ripening cultivar, Ilpum. The authors identified regional specificity for cultivation among five areas in Korea which were characterized by polishing degree and cultivar type. Finally, the current study proposes a possible linkage of the region-specific metabolic signatures to soil texture and total rainfall. In addition, we found tryptophan metabolites that implied the potential for microbe-host interactions that may influence crop metabolites.

Discrimination of the Geographical Origin of Soybeans Using NMR-Based Metabolomics.

With the increase in soybean trade between countries, the intentional mislabeling of the origin of soybeans has become a serious problem worldwide. In this study, metabolic profiling of soybeans from the Republic of Korea and China was performed by nuclear magnetic resonance (NMR) spectroscopy coupled with multivariate statistical analysis to predict the geographical origin of soybeans. The optimal orthogonal partial least squares-discriminant analysis (OPLS-DA) model was obtained using total area normalization and unit variance (UV) scaling, without applying the variable influences on projection (VIP) cut-off value, resulting in 96.9% sensitivity, 94.4% specificity, and 95.6% accuracy in the leave-one-out cross validation (LOO-CV) test for discriminating between Korean and Chinese soybeans. Soybeans from the northeastern, middle, and southern regions of China were successfully differentiated by standardized area normalization and UV scaling with a VIP cut-off value of 1.0, resulting in 100% sensitivity, 91.7%-100% specificity, and 94.4%-100% accuracy in a LOO-CV test. The methods employed in this study can be used to obtain essential information for the authentication of soybean samples from diverse geographical locations in future studies.

ARS2 Plays Diverse Roles in DNA Damage Response, Fungal Development, and Pathogenesis in the Plant Pathogenic Fungus Fusarium graminearum.

Arsenite-resistance protein 2 (Ars2) is an important nuclear protein involved in various RNA metabolisms in animals and plants, but no Ars2 ortholog has been studied in filamentous fungi. Although it is an essential gene in most model eukaryotes, FgARS2 null mutants were viable in the plant pathogenic fungus Fusarium graminearum. The deletion of FgARS2 resulted in pleiotropic defects in various fungal developmental processes. Fgars2 mutants were irregular in nuclear division, and conidial germination was significantly retarded, causing the fungus to manifest its hypersensitive phenotypes under DNA damage stress. While FgARS2 deletion caused abnormal morphologies of ascospores and defective ascospore discharge, our data revealed that FgARS2 was not closely involved in small-non-coding RNA production in F. graminearum. The dominant nuclear localization of FgArs2-green fluorescent proteins (GFP) and abnormal nuclear division in FgARS2 deletion mutant implicated that FgArs2 functions in the nucleus. Intriguingly, we found that FgArs2 established a robust physical interaction with the cap binding complex (CBC) to form a tertiary complex CBC-Ars2 (CBCA), and disruption of any CBCA complex subunit drastically attenuated the virulence of F. graminearum. The results of the study indicate that Ars2 regulates fungal development, stress response, and pathogenesis via interaction with CBC in F. graminearum and provide a novel insight into understanding of the biological functions of Ars2 in filamentous fungi.

Comparative study on metabolic changes of Aspergillus oryzae isolated from fermented foods according to culture conditions.

Aspergillus oryzae is an important microbial starter for making diverse fermented foods due to its high hydrolytic enzyme activities. In this study, two strains of A. oryzae (AOB/AOK) with different activities of hydrolytic enzymes, such as α-amylase, protease and lipase, were cultured under various conditions of temperature, fermentation time, and initial pH. Comparative mass spectrometry (MS) based metabolomic analysis was performed to obtain primary metabolites and secondary volatile metabolite data sets. In the results of partial least square-discriminant analysis (PLS-DA), fatty acids and volatile metabolites derived from fatty acids and amino acids mainly contributed to AOK with higher protease and lipase activities, whereas carbohydrate-derived volatiles, sugars and sugar alcohols were related to main metabolites of AOB with higher α-amylase activity. The temperature and initial pH were critical factors for the generation of primary metabolites and secondary volatile metabolites, such as organic acids, fatty acids-derived volatiles, and some amino acids, in both A. oryzae strains. This study demonstrated that the specific culture conditions were closely linked to the formation of primary metabolites and secondary volatile metabolites of A. oryzae.

Effects of pH and Cultivation Time on the Formation of Styrene and Volatile Compounds by Penicillium expansum.

Styrene can be formed by the microbial metabolism of bacteria and fungi. In our previous study, styrene was determined as a spoilage marker of Fuji apples decayed by Penicillium expansum, which is responsible for postharvest diseases. In the present study, P. expansum was cultivated in potato dextrose broth added with phenylalanine-which is a precursor of styrene-using different initial pH values and cultivation times. Volatile compounds were extracted and analyzed using gas chromatography-mass spectrometry (GC-MS) combined with stir-bar sorptive extraction. The 76 detected volatile compounds included 3-methylbutan-1-ol, 3-methyl butanal, oct-1-en-3-ol, geosmin, nonanal, hexanal, and γ-decalactone. In particular, the formation of 10 volatile compounds derived from phenylalanine (including styrene and 2-phenylethanol) showed different patterns according to pH and the cultivation time. Partial least square-discriminant analysis (PLS-DA) plots indicated that the volatile compounds were affected more by pH than by the cultivation time. These results indicated that an acidic pH enhances the formation of styrene and that pH could be a critical factor in the production of styrene by P. expansum. This is the first study to analyze volatile compounds produced by P. expansum according to pH and cultivation time and to determine their effects on the formation of styrene.

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.

Discrimination and prediction of the origin of Chinese and Korean soybeans using Fourier transform infrared spectrometry (FT-IR) with multivariate statistical analysis.

The ability to determine the origin of soybeans is an important issue following the inclusion of this information in the labeling of agricultural food products becoming mandatory in South Korea in 2017. This study was carried out to construct a prediction model for discriminating Chinese and Korean soybeans using Fourier-transform infrared (FT-IR) spectroscopy and multivariate statistical analysis. The optimal prediction models for discriminating soybean samples were obtained by selecting appropriate scaling methods, normalization methods, variable influence on projection (VIP) cutoff values, and wave-number regions. The factors for constructing the optimal partial-least-squares regression (PLSR) prediction model were using second derivatives, vector normalization, unit variance scaling, and the 4000-400 cm-1 region (excluding water vapor and carbon dioxide). The PLSR model for discriminating Chinese and Korean soybean samples had the best predictability when a VIP cutoff value was not applied. When Chinese soybean samples were identified, a PLSR model that has the lowest root-mean-square error of the prediction value was obtained using a VIP cutoff value of 1.5. The optimal PLSR prediction model for discriminating Korean soybean samples was also obtained using a VIP cutoff value of 1.5. This is the first study that has combined FT-IR spectroscopy with normalization methods, VIP cutoff values, and selected wave-number regions for discriminating Chinese and Korean soybeans.

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.

Comprehensive analysis of fungal diversity and enzyme activity in nuruk, a Korean fermenting starter, for acquiring useful fungi.

Nuruk is a fermenting starter that is involved in the production of alcoholic beverages, and has been used in South Korea for a very long time. To analyze the fungal diversity, we collected a total of 59 nuruk samples from several companies and persons in 2013 to 2014, and obtained 364 isolates. All of the single isolated fungi were identified, both morphologically and molecularly, based on the sequences of ribosomal RNA gene [18S, ITS1-5.8S-ITS2, and 26S (D1/D2 region)]. In 46 nuruk samples out of 59 (78%), Saccharomycopsis fibuligera, a dimorphic yeast, was most frequently isolated. Among the filamentous fungi, Aspergillus and Lichtheimia were found in more than 50% of the samples with lower colony forming unit (CFU/g of sample) than those of yeasts. The yeasts S. fibuligera and Wickerhamomyces anomalus were counted with maximum 1.3-1.8 × 108 CFU/g. Among Mucorales fungi, Lichtheimia and Mucor were isolated in much higher numbers than Rhizopus and Rhizomucor. Overall, the home-made nuruks tend to contain more diverse filamentous fungi than the commercial nuruks. To acquire industrially useful filamentous fungi and yeasts, we analyzed the enzyme activities of α-amylase, glucoamylase and acid protease associated with brewing properties for 131 strains. Aspergillus oryzae and S. fibuligera had high α- and glucoamylase activities and most isolates of Lichtheimia ramosa had high acid protease activity. For further applications, 27 fungal strains were chosen based on isolation frequencies from nuruk, and the ability to produce useful enzyme.

Comparative analysis of nonvolatile and volatile metabolites in Lichtheimia ramosa cultivated in different growth media.

Lichtheimia ramosa is one of the predominant filamentous fungi in Korean traditional nuruk. The nonvolatile and volatile metabolites of L. ramosa cultivated in three growth media: complete medium (CM), potato dextrose broth (PDB), and sabouraud dextrose broth (SDB), were investigated and compared. Among nonvolatile metabolites, serine, lysine, and ornithine increased in CM and PDB cultivated with L. ramosa during the exponential phase. In addition, glucose level increased in CM whereas decreased in PDB and SDB. The major volatile metabolites in the extract samples were acetic acid, ethanol, 3-methyl-2-buten-1-ol, 2-phenylethanol, ethylacetate, 2-furaldehyde, 5-(hydroxymethyl)-2-furaldehyde, 2,3-dihydro-3,5,-dihydroxy-6-methyl-4H-pyran-4-one, and α-humulene. In particular, the levels of volatile metabolites related to makgeolli (e.g., acetic acid, ethanol, and ethyl acetate) were highest in extracts cultivated in CM. On the other hand, the level of 2-phenylethanol was relatively higher in PDB and SDB, possibly due to there being more phenylalanine present in the biomass sample in media.

Whole-genome de novo sequencing, combined with RNA-Seq analysis, reveals unique genome and physiological features of the amylolytic yeast Saccharomycopsis fibuligera and its interspecies hybrid.

BACKGROUND: Genomic studies on fungal species with hydrolytic activity have gained increased attention due to their great biotechnological potential for biomass-based biofuel production. The amylolytic yeast Saccharomycopsis fibuligera has served as a good source of enzymes and genes involved in saccharification. Despite its long history of use in food fermentation and bioethanol production, very little is known about the basic physiology and genomic features of S. fibuligera. RESULTS: We performed whole-genome (WG) de novo sequencing and complete assembly of S. fibuligera KJJ81 and KPH12, two isolates from wheat-based Nuruk in Korea. Intriguingly, the KJJ81 genome (~38 Mb) was revealed as a hybrid between the KPH12 genome (~18 Mb) and another unidentified genome sharing 88.1% nucleotide identity with the KPH12 genome. The seven chromosome pairs of KJJ81 subgenomes exhibit highly conserved synteny, indicating a very recent hybridization event. The phylogeny inferred from WG comparisons showed an early divergence of S. fibuligera before the separation of the CTG and Saccharomycetaceae clades in the subphylum Saccharomycotina. Reconstructed carbon and sulfur metabolic pathways, coupled with RNA-Seq analysis, suggested a marginal Crabtree effect under high glucose and activation of sulfur metabolism toward methionine biosynthesis under sulfur limitation in this yeast. Notably, the lack of sulfate assimilation genes in the S. fibuligera genome reflects a unique phenotype for Saccharomycopsis clades as natural sulfur auxotrophs. Extended gene families, including novel genes involved in saccharification and proteolysis, were identified. Moreover, comparative genome analysis of S. fibuligera ATCC 36309, an isolate from chalky rye bread in Germany, revealed that an interchromosomal translocation occurred in the KPH12 genome before the generation of the KJJ81 hybrid genome. CONCLUSIONS: The completely sequenced S. fibuligera genome with high-quality annotation and RNA-Seq analysis establishes an important foundation for functional inference of S. fibuligera in the degradation of fermentation mash. The gene inventory facilitates the discovery of new genes applicable to the production of novel valuable enzymes and chemicals. Moreover, as the first gapless genome assembly in the genus Saccharomycopsis including members with desirable traits for bioconversion, the unique genomic features of S. fibuligera and its hybrid will provide in-depth insights into fungal genome dynamics as evolutionary adaptation.