Characterization of a new Saccharomyces cerevisiae isolated from banana stems and its mutant with l-leucine accumulation for awamori brewing.

We isolated a new strain of the yeast Saccharomyces cerevisiae, 35a14, from banana stems in Okinawa. This strain did not belong to any industrial yeast groups in a phylogenetic tree and produced high levels of alcohol. Furthermore, awamori, an Okinawa's traditional distilled alcoholic beverage, brewed with an l-leucine overproducing mutant derived from 35a14 showed a high concentration of isoamyl acetate.

Draft Genome Sequence of Saccharomyces cerevisiae Strain Awamori Number 101, Commonly Used to Make Awamori, a Traditional Spirit, in Okinawa, Japan.

We report here the draft genome sequence for Saccharomyces cerevisiae strain Awamori number 101, an industrial strain used for producing awamori, a distilled alcohol beverage. It was constructed by assembling the short reads obtained by next-generation sequencing. The 315 contigs constitute an 11.5-Mbp genome sequence encoding 6,185 predicted proteins.

Whole-Genome Sequence of Monascus purpureus GB-01, an Industrial Strain for Food Colorant Production.

We report the draft genome sequence of Monascus purpureus GB-01, an industrial strain used as a food colorant. De novo assembly of long reads resulted in 121 chromosomal contigs and 1 mitochondrial contig, and sequencing errors were corrected by paired-end short reads. This genome sequence will provide useful information for azaphilone pigments and mycotoxin citrinin biosynthesis.

Characterization of a New Saccharomyces cerevisiae Isolated From Hibiscus Flower and Its Mutant With L-Leucine Accumulation for Awamori Brewing.

Since flavors of alcoholic beverages produced in fermentation process are affected mainly by yeast metabolism, the isolation and breeding of yeasts have contributed to the alcoholic beverage industry. To produce awamori, a traditional spirit (distilled alcoholic beverage) with unique flavors made from steamed rice in Okinawa, Japan, it is necessary to optimize yeast strains for a diversity of tastes and flavors with established qualities. Two categories of flavors are characteristic of awamori; initial scented fruity flavors and sweet flavors that arise with aging. Here we isolated a novel strain of Saccharomyces cerevisiae from hibiscus flowers in Okinawa, HC02-5-2, that produces high levels of alcohol. The whole-genome information revealed that strain HC02-5-2 is contiguous to wine yeast strains in a phylogenic tree. This strain also exhibited a high productivity of 4-vinyl guaiacol (4-VG), which is a precursor of vanillin known as a key flavor of aged awamori. Although conventional awamori yeast strain 101-18, which possesses the FDC1 pseudogene does not produce 4-VG, strain HC02-5-2, which has the intact PAD1 and FDC1 genes, has an advantage for use in a novel kind of awamori. To increase the contents of initial scented fruity flavors, such as isoamyl alcohol and isoamyl acetate, we attempted to breed strain HC02-5-2 targeting the L-leucine synthetic pathway by conventional mutagenesis. In mutant strain T25 with L-leucine accumulation, we found a hetero allelic mutation in the LEU4 gene encoding the Gly516Ser variant α-isopropylmalate synthase (IPMS). IPMS activity of the Gly516Ser variant was less sensitive to feedback inhibition by L-leucine, leading to intracellular L-leucine accumulation. In a laboratory-scale test, awamori brewed with strain T25 showed higher concentrations of isoamyl alcohol and isoamyl acetate than that brewed with strain HC02-5-2. Such a combinatorial approach to yeast isolation, with whole-genome analysis and metabolism-focused breeding, has the potentials to vary the quality of alcoholic beverages.

Genome sequence of Aspergillus luchuensis NBRC 4314.

Awamori is a traditional distilled beverage made from steamed Thai-Indica rice in Okinawa, Japan. For brewing the liquor, two microbes, local kuro (black) koji mold Aspergillus luchuensis and awamori yeast Saccharomyces cerevisiae are involved. In contrast, that yeasts are used for ethanol fermentation throughout the world, a characteristic of Japanese fermentation industries is the use of Aspergillus molds as a source of enzymes for the maceration and saccharification of raw materials. Here we report the draft genome of a kuro (black) koji mold, A. luchuensis NBRC 4314 (RIB 2604). The total length of nonredundant sequences was nearly 34.7 Mb, comprising approximately 2,300 contigs with 16 telomere-like sequences. In total, 11,691 genes were predicted to encode proteins. Most of the housekeeping genes, such as transcription factors and N-and O-glycosylation system, were conserved with respect to Aspergillus niger and Aspergillus oryzae An alternative oxidase and acid-stable α-amylase regarding citric acid production and fermentation at a low pH as well as a unique glutamic peptidase were also found in the genome. Furthermore, key biosynthetic gene clusters of ochratoxin A and fumonisin B were absent when compared with A. niger genome, showing the safety of A. luchuensis for food and beverage production. This genome information will facilitate not only comparative genomics with industrial kuro-koji molds, but also molecular breeding of the molds in improvements of awamori fermentation.

Draft Genome Sequence of Lactobacillus gorillae Strain KZ01T, Isolated from a Western Lowland Gorilla.

Here, we report the draft genome sequence of Lactobacillus gorillae strain KZ01(T) isolated from a western lowland gorilla (Gorilla gorilla gorilla). This genome sequence will be helpful for the comparative genomics between human and nonhuman primate-associated Lactobacillus.

Isolation and characterization of awamori yeast mutants with L-leucine accumulation that overproduce isoamyl alcohol.

Awamori shochu is a traditional distilled alcoholic beverage made from steamed rice in Okinawa, Japan. Although it has a unique aroma that is distinguishable from that of other types of shochu, no studies have been reported on the breeding of awamori yeasts. In yeast, isoamyl alcohol (i-AmOH), known as the key flavor of bread, is mainly produced from α-ketoisocaproate in the pathway of L-leucine biosynthesis, which is regulated by end-product inhibition of α-isopropylmalate synthase (IPMS). Here, we isolated mutants resistant to the L-leucine analog 5,5,5-trifluoro-DL-leucine (TFL) derived from diploid awamori yeast of Saccharomyces cerevisiae. Some of the mutants accumulated a greater amount of intracellular L-leucine, and among them, one mutant overproduced i-AmOH in awamori brewing. This mutant carried an allele of the LEU4 gene encoding the Ser542Phe/Ala551Val variant IPMS, which is less sensitive to feedback inhibition by L-leucine. Interestingly, we found that either of the constituent mutations (LEU4(S542F) and LEU4(A551V)) resulted in the TFL tolerance of yeast cells and desensitization to L-leucine feedback inhibition of IPMS, leading to intracellular L-leucine accumulation. Homology modeling also suggested that L-leucine binding was drastically inhibited in the Ser542Phe, Ala551Val, and Ser542Phe/Ala551Val variants due to steric hindrance in the cavity of IPMS. As we expected, awamori yeast cells expressing LEU4(S542F), LEU4(A551V), and LEU4(S542F/A551V) showed a prominent increase in extracellular i-AmOH production, compared with that of cells carrying the vector only. The approach described here could be a practical method for the breeding of novel awamori yeasts to expand the diversity of awamori taste and flavor.

The novel helicase helG (DHX30) is expressed during gastrulation in mice and has a structure similar to a human DExH box helicase.

The gene trap method for embryonic stem cells is an efficient method for identifying new genes that are involved in development. Using this method, we identified a novel gene called helicase family gene related to gastrulation (helG). Helicase family proteins regulate many systems in the body that are related to cell survival. HelG encodes a protein of 137 kDa, which contains a DExH helicase motif that is now named DHX30. HelG is strongly expressed in neural cells (ie, in the headfold, neural plate, neural tube, and brain) and somites during embryogenesis. Growing homozygous mutant embryos have neither differentiated somites nor brains. In these mutants, development was retarded by embryonic day 7.5 (E7.5), and the mutants died at E9.5. After the purification of HelG, an untwisting experiment was performed to confirm the helicase activity of HelG for DNA in vitro. We report for the first time that a helicase family gene is required for differentiation during embryogenesis; this gene might interact with polynucleotides to regulate some genes that are important for early development and has a structure similar to that of a human DExH box helicase.

Draft Genome Sequence of Saccharomyces cerevisiae NAM34-4C, a Lactic Acid-Assimilating Industrial Yeast Strain.

We determined the genome sequence of industrial Saccharomyces cerevisiae strain NAM34-4C, which would be useful for bioethanol production. The approximately 11.5-Mb draft genome sequence of NAM34-4C will provide remarkable insights into metabolic engineering for effective production of bioethanol from biomass.

Draft Genome Sequence of Saccharomyces cerevisiae IR-2, a Useful Industrial Strain for Highly Efficient Production of Bioethanol.

We sequenced the genome of Saccharomyces cerevisiae IR-2, which is a diploid industrial strain with flocculation activity and the ability to efficiently produce bioethanol. The approximately 11.4-Mb draft genome information provides useful insights into metabolic engineering for the production of bioethanol from biomass.

Comparative genome analysis between Aspergillus oryzae strains reveals close relationship between sites of mutation localization and regions of highly divergent genes among Aspergillus species.

Aspergillus oryzae has been utilized for over 1000 years in Japan for the production of various traditional foods, and a large number of A. oryzae strains have been isolated and/or selected for the effective fermentation of food ingredients. Characteristics of genetic alterations among the strains used are of particular interest in studies of A. oryzae. Here, we have sequenced the whole genome of an industrial fungal isolate, A. oryzae RIB326, by using a next-generation sequencing system and compared the data with those of A. oryzae RIB40, a wild-type strain sequenced in 2005. The aim of this study was to evaluate the mutation pressure on the non-syntenic blocks (NSBs) of the genome, which were previously identified through comparative genomic analysis of A. oryzae, Aspergillus fumigatus, and Aspergillus nidulans. We found that genes within the NSBs of RIB326 accumulate mutations more frequently than those within the SBs, regardless of their distance from the telomeres or of their expression level. Our findings suggest that the high mutation frequency of NSBs might contribute to maintaining the diversity of the A. oryzae genome.

Molecular biological researches of Kuro-Koji molds, their classification and safety.

To assess the position of Kuro-Koji molds in black Aspergillus, we performed sequence analysis of approximately 2500 nucleotides of partial gene fragments, such as histone 3, on a total of 57 Aspergillus strains, including Aspergillus kawachii NBRC 4308, 12 Kuro-Koji molds isolated from awamori breweries in Japan, Aspergillus niger ATCC 1015, and A. tubingensis ATCC10550. Sequence results showed that all black Aspergillus strains could be classified into 3 types, type N which includes A. niger ATCC 1015, type T which includes A. tubingensis ATCC 10550, and type L which includes A. kawachii NBRC 4308. Phylogenetic analysis showed these three types belong to different clusters. All 12 Kuro-Koji molds isolated from awamori breweries were classified as type L, thus we concluded type L represents the industrial Kuro-Koji molds. We found all type L strains lack the An15g07920 gene which is required for ochratoxin A biosynthesis in black Aspergillus. This sequence is present in the genome of A. niger CBS 513.88 and has homology to the polyketide synthase fragment of A. ochraceus which is involved in ochratoxin A biosynthesis. Based on the industrial importance and the safety of Kuro-Koji molds, we propose to classify the type L strains as Aspergillus luchuensis, as initially reported by Dr. Inui.

Application of random amplified polymorphic DNA (RAPD) analysis coupled with microchip electrophoresis for high-resolution identification of Monascus strains.

Monascus fungi are commonly used for a variety of food products in Asia, and are also known to produce some biologically active compounds. Since the use of Monascus is expected to increase in food industries, strain-level identification and management of Monascus will be needed in the near future. In the present study, random amplified polymorphic DNA (RAPD) analysis coupled with microchip electrophoresis was applied for this purpose. Evaluations of the analysis stability revealed that reproducible results could be obtained, although template DNA fragmentation could influence the resulting RAPD pattern. RAPD analysis using 15 Monascus strains consisting of four species, M. ruber, M. pilosus, M. purpureus, and M. kaoliang showed that each strain generated a unique RAPD pattern, which allows strain-level identification of Monascus. In addition, the phylogenetic tree constructed from RAPD patterns reflected M. ruber-M. pilosus and M. purpureus-M. kaoliang clusters inferred from both ITS and beta-tubulin gene sequences, which indicated that the RAPD pattern could reflect their phylogenetic traits to a certain extent. On the other hand, RAPD analysis did not support the monophyletic clustering of the four Monascus species used in this study, which suggests the necessity of reexamination of species boundaries in Monascus.

Red yeast rice fermentation by selected Monascus sp. with deep-red color, lovastatin production but no citrinin, and effect of temperature-shift cultivation on lovastatin production.

Monascus pilosus NBRC4520 was selected for functional fermented food inoculation for its high lovastatin and low citrinin production with a deep-red color. For koji (mold rice) with high lovastatin production, separation of the growth phase and lovastatin production phase by shifting the temperature from 30 to 23 degrees C increased lovastatin production by nearly 20 times compared to temperature-constant cultivation. In addition, citrinin was not produced even in the lovastatin production phase, although the pigment was increased. With temperature-shift cultivation, 225 microg lovastatin/g dry koji was produced in 14 days without citrinin.