Importance and mechanisms of S-adenosylmethionine and folate accumulation in sake yeast.

Sake yeasts have a range of brewing characteristics that are particularly beneficial for sake making including high ethanol fermentability, high proliferative capacity at low temperatures, lactic acid tolerance, and high ester productivity. On the other hand, sake yeasts also accumulate a diverse range of functional components. For example, significantly greater accumulation of S-adenosylmethionine (SAM), a compound that plays important regulatory roles in a range of biological processes as a major donor of methyl groups, occurs in sake yeasts compared to other microorganisms. Significantly greater accumulation of folate, a bioactive water-soluble vitamin (vitamin B9), also occurs in sake yeasts compared to laboratory yeasts, and the methyl group on SAM is supplied by folate. Accordingly, fully characterizing 'sake yeast identity' requires detailed understanding of the mechanisms underlying both the nutritional characteristics (functional components) and the brewing characteristics in sake yeasts. Therefore, this mini-review focuses on the accumulation of SAM and folate in sake yeast including descriptions of the genes known to contribute to SAM and folate accumulation and the underlying mechanisms.

The sake yeast YHR032W/ERC1 allele contributes to the regulation of the tetrahydrofolate content in the folate synthetic pathway in sake yeast strains.

To elucidate the mechanism underlying tetrahydrofolate (THF) accumulation in sake yeast strains compared with that in laboratory yeast strains, we performed a quantitative trait locus (QTL) analysis. The results revealed that the sake yeast ERC1 allele contributes to an increase in the ratio of THF to the total folate content in sake yeast.

Breeding of a cordycepin-resistant and adenosine kinase-deficient sake yeast strain that accumulates high levels of S-adenosylmethionine.

Adenosine kinase (ADO1)-deficient mutants can be obtained from cordycepin-resistant strains, and the disruption of ADO1 causes S-adenosylmethionine (SAM) accumulation. To breed a high-SAM-accumulating yeast strain without genetic manipulation for industrial purposes, we bred a cordycepin-resistant strain using sake yeast kyokai No. 9 as the parent strain with a mutation in adenosine kinase (ADO1) and acquired high-SAM-accumulating strain. In the bred strain (NY9-10), a single mutation (T258I) was present in the ADO1, and this mutation site is an ATP binding site and is highly conserved during evolution. Moreover, it was suggested that high accumulation of SAM and cordycepin resistance in NY9-10 was due to functional deficiency of ADO1 by this mutation. This strain is not a genetically-modified organism and can be employed for use in the food and medicine industry such as mass production and sake making.

A genetic method to enhance the accumulation of S-adenosylmethionine in yeast.

S-Adenosylmethionine (SAM) is a key component of sulphur amino acid metabolism in living organisms and is synthesised from methionine and adenosine triphosphate by methionine adenosyltransferase. This molecule serves as the main biological methyl donor due to its active methylthio ether group. Notably, SAM has shown beneficial effects in clinical trials for the treatment of alcoholic liver disease, depression and joint pain. Due to the high potential value of SAM, current research efforts are attempting to develop a more rapid, cost-effective and higher yielding SAM production method than the conventional production system. In this mini-review, we describe the previously reported yeast gene that contributes to SAM accumulation by overexpression, mutation or deletion and summarise the genetic approach for the production of SAM in large industrial quantities.

Sake yeast YHR032W/ERC1 haplotype contributes to high S-adenosylmethionine accumulation in sake yeast strains.

Sake yeasts are ideally suited for sake making, producing higher levels of ethanol, proliferating at lower temperatures, and producing greater levels of various aromatic components and nutrients than laboratory yeasts. To elucidate the mechanism underlying S-adenosylmethionine (SAM) accumulation in sake yeast strains compared with that in laboratory yeast strains, we performed quantitative trait locus (QTL) analysis and identified a significant QTL on chromosome VIII. Of the 165 genes mapped at 49.8 cM from the left-end DNA marker of chromosome VIII, we focused on the YHR032W/ERC1 gene, encoding a member of the multi-drug and toxin extrusion family having antiporter activity and involved in SAM accumulation and ethionine resistance. Expression of the sake yeast ERC1 haplotype (K7ERC1) in a low- and high-copy number plasmid BYΔerc1 resulted in intracellular SAM accumulation, whereas expression of the laboratory yeast ERC1 haplotype (XERC1) did not. Comparison between DNA sequences of K7ERC1 and XERC1 revealed three amino acid substitutions: S51N, V263I, and N545I. Site-directed mutagenesis revealed that the N545I frameshift mutation was responsible for the K7ERC1 phenotype. These results indicate that K7ERC1 contributes to SAM accumulation in sake yeast strains.

Stimulating S-adenosyl-l-methionine synthesis extends lifespan via activation of AMPK.

Dietary restriction (DR), such as calorie restriction (CR) or methionine (Met) restriction, extends the lifespan of diverse model organisms. Although studies have identified several metabolites that contribute to the beneficial effects of DR, the molecular mechanism underlying the key metabolites responsible for DR regimens is not fully understood. Here we show that stimulating S-adenosyl-l-methionine (AdoMet) synthesis extended the lifespan of the budding yeast Saccharomyces cerevisiae The AdoMet synthesis-mediated beneficial metabolic effects, which resulted from consuming both Met and ATP, mimicked CR. Indeed, stimulating AdoMet synthesis activated the universal energy-sensing regulator Snf1, which is the S. cerevisiae ortholog of AMP-activated protein kinase (AMPK), resulting in lifespan extension. Furthermore, our findings revealed that S-adenosyl-l-homocysteine contributed to longevity with a higher accumulation of AdoMet only under the severe CR (0.05% glucose) conditions. Thus, our data uncovered molecular links between Met metabolites and lifespan, suggesting a unique function of AdoMet as a reservoir of Met and ATP for cell survival.

Heterologous production of horseradish peroxidase C1a by the basidiomycete yeast Cryptococcus sp. S-2 using codon and signal optimizations.

In the present study, we attempted to improve the production of recombinant horseradish peroxidase C1a (HRP-C1a; a heme-binding protein) by Cryptococcus sp. S-2. Both native and codon-optimized HRP-C1a genes were expressed under the control of a high-level expression promoter. When the HRP-C1a gene with native codons was expressed, poly(A) tails tended to be added within the coding region, producing truncated messenger RNAs (mRNAs) that lacked the 3' ends. Codon optimization prevented polyadenylation within the coding region and increased both the mRNA and protein levels of active HRP-C1a. To improve secretion of the recombinant protein, we tested five types of N-terminal signal peptide (NTP). These included the native HRP-C1a NTP (C1a-NTP), short and long xylanase secretion signals (X1-NTP and X2-NTP), cutinase signal (C-NTP), and amylase signal (A-NTP), with and without a C-terminal propeptide (CTP). X2-NTP without CTP resulted in the highest HRP-C1a secretion into the culture medium. HRP-C1a secretion was further increased by using xylose fed-batch fermentation. The production of HRP-C1a in this study was 2.7 and 15 times higher than the production reported in previous studies that used insect cell and Pichia expression systems, respectively.

Single nucleotide polymorphisms of PAD1 and FDC1 show a positive relationship with ferulic acid decarboxylation ability among industrial yeasts used in alcoholic beverage production.

Among industrial yeasts used for alcoholic beverage production, most wine and weizen beer yeasts decarboxylate ferulic acid to 4-vinylguaiacol, which has a smoke-like flavor, whereas sake, shochu, top-fermenting, and bottom-fermenting yeast strains lack this ability. However, the factors underlying this difference among industrial yeasts are not clear. We previously confirmed that both PAD1 (phenylacrylic acid decarboxylase gene, YDR538W) and FDC1 (ferulic acid decarboxylase gene, YDR539W) are essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae. In the present study, single nucleotide polymorphisms (SNPs) of PAD1 and FDC1 in sake, shochu, wine, weizen, top-fermenting, bottom-fermenting, and laboratory yeast strains were examined to clarify the differences in ferulic acid decarboxylation ability between these types of yeast. For PAD1, a nonsense mutation was observed in the gene sequence of standard top-fermenting yeast. Gene sequence analysis of FDC1 revealed that sake, shochu, and standard top-fermenting yeasts contained a nonsense mutation, whereas a frameshift mutation was identified in the FDC1 gene of bottom-fermenting yeast. No nonsense or frameshift mutations were detected in laboratory, wine, or weizen beer yeast strains. When FDC1 was introduced into sake and shochu yeast strains, the transformants exhibited ferulic acid decarboxylation activity. Our findings indicate that a positive relationship exists between SNPs in PAD1 and FDC1 genes and the ferulic acid decarboxylation ability of industrial yeast strains.

Treatment of, and Candida utilis biomass production from shochu wastewater; the effects of maintaining a low pH on DOC removal and feeding cultivation on biomass production.

Shochu wastewater (SW; alcoholic distillery wastewater) contains large amounts of organic compounds (25,000 - 60,000 COD mg/L), nitrogen (1,000 - 6,000 T-N mg/L), and phosphorus (500 - 1,000 T-P mg/L). Despite its high nutrient content, SW is highly perishable, which limits its utilization for animal feed and fertilizer. Therefore, SW is mainly treated by methane fermentation. On the other hand, a feed yeast, Candida utilis, can utilize various organic compounds and be utilized as a yeast extract source and animal feed. We previously bred a mutant, C. utilis UNA1, that accumulates a large amount of nitrogen. Here, we investigated the use of C. utilis UNA1 to treat highly concentrated SW. With fed-batch cultivation using a 5-L jar fermenter, controlling pH at 5.0 with H2SO4, 62.9% of DOC, 38.4% of DTN, and 44.5% of DTP were stably removed from non-diluted barley shochu wastewater (BSW), and about 16.7 kg of freeze-dried yeast biomass was obtained. The yeast sludge biomass generated from BSW contains about 60% crude protein. Furthermore, using H2SO4 to control pH increased the sulfur content of wastewater, which increased the methionine composition of yeast sludge biomass.

Genome-wide screening to study breeding methods to improve the nitrogen accumulation ability of yeast without gene recombinant techniques.

To remove nitrogen efficiently from high-concentration organic wastewater, we studied breeding methods using Saccharomyces cerevisiae as a model yeast with improved nitrogen accumulation ability. By DNA microarray analysis under various nitrogen concentrations with two nitrogen sources (peptone and L-asparagine), we obtained 295 commonly overexpressed (over 2-fold) genes and 283 commonly underexpressed (under one-half) genes under nitrogen-starvation conditions. We speculated that overexpression or underexpression recombination of some of these genes might enhance nitrogen uptake. Because a complete collection of nonessential gene deletion strains had been created, we investigated the nitrogen accumulation profiles of underexpressed gene deletion strains. From 256 nonessential gene deletion strains, three (URE2, SNO1, and AVT3) were selected. Strain SUD2 (ure2Δ::kanMX4) improved by 1.2-fold total nitrogen per cell (TN/OD660) as compared to the parent strain, S288c. Positive selection of methylamine-resistant mutants to obtain URE2 mutants was useful for improving nitrogen accumulation ability without recombinant techniques.

Fusion of cellulose binding domain from Trichoderma reesei CBHI to Cryptococcus sp. S-2 cellulase enhances its binding affinity and its cellulolytic activity to insoluble cellulosic substrates.

Cryptococcus sp. S-2 carboxymethyl cellulase (CSCMCase) is active in the acidic pH and lacks a binding domain. The absence of the binding domain makes the enzyme inefficient against insoluble cellulosic substrates. To enhance its binding affinity and its cellulolytic activity to insoluble cellulosic substrates, cellulose binding domain (CBD) of cellobiohydrolase I (CBHI) from Trichoderma reesei belonging to carbohydrate binding module (CBM) family 1 was fused at the C-terminus of CSCMCase. The constructed fusion enzymes (CSCMCase-CBD and CSCMCase-2CBD) were expressed in a newly recombinant expression system of Cryptococcus sp. S-2, purified to homogeneity, and then subject to detailed characterization. The recombinant fusion enzymes displayed optimal pH similar to those of the native enzyme. Compared with rCSCMCase, the recombinant fusion enzymes had acquired an increased binding affinity to insoluble cellulose and the cellulolytic activity toward insoluble cellulosic substrates (SIGMACELL(®) and Avicel) was higher than that of native enzyme, confirming the presence of CBDs improve the binding and the cellulolytic activity of CSCMCase on insoluble substrates. This attribute should make CSCMCase an attractive applicant for various application.

Adenosine kinase-deficient mutant of Saccharomyces cerevisiae accumulates S-adenosylmethionine because of an enhanced methionine biosynthesis pathway.

To isolate an S-adenosylmethionine (SAM)-accumulating yeast strain and to develop a more efficient method of producing SAM, we screened methionine-resistant strains using the yeast deletion library of budding yeast and isolated 123 strains. The SAM content in 81 of the 123 strains was higher than that in the parental strain BY4742. We identified ADO1 encoding adenosine kinase as one of the factors participating in high SAM accumulation. The X∆ado1 strain that was constructed from the X2180-1A strain (MAT a, ATCC 26786) could accumulate approximately 30-fold (18 mg/g dry cell weight) more SAM than the X2180-1A strain in yeast extract peptone dextrose medium. Furthermore, we attempted to identify the molecular basis underlying the differences in SAM accumulation between X∆ado1 and X2180-1A strains. DNA microarray analysis revealed that the genes involved in the methionine biosynthesis pathway, phosphate metabolism, and hexose transport were mainly overexpressed in the X∆ado1 strain compared with the X2180-1A strain. We also determined the levels of various metabolites involved in the methionine biosynthesis pathway and found increased content of SAM, tetrahydrofolate (THF), inorganic phosphate, polyphosphoric acid, and S-adenosylhomocysteine in the X∆ado1 strain. In contrast, the content of 5-methyl-THF, homocysteine, glutathione, and adenosine was decreased. These results indicated that the ∆ado1 strain could accumulate SAM because of preferential activation of the methionine biosynthesis pathway.

Comparison of laccase production levels in Pichia pastoris and Cryptococcus sp. S-2.

The heterologous expression of the laccase gene from Trametes versicolor and Gaeumannomyces graminis was evaluated in the yeasts Pichia pastoris and Cryptococcus sp. S-2. The expression levels of both laccase genes in Cryptococcus sp. S-2 were considerably higher than those in P. pastoris. The codon usage of Cryptococcus sp. S-2 as well as the GC content were similar to those of T. versicolor and G. graminis. These results suggest that using a host with a similar codon usage for the expressed gene may improve protein expression. The use of Cryptococcus sp. S-2 as a host may be advantageous for the heterologous expression of genes with high GC content. Moreover, this yeast provides the same advantages as P. pastoris for the production of recombinant proteins, such as growth on minimal medium, capacity for high-density growth during fermentation, and capability for post-translational modifications. Therefore, we propose that Cryptococcus sp. S-2 be used as an expression host to improve enzyme production levels when other hosts have not yielded good results.

Modified Cre-loxP recombination in Aspergillus oryzae by direct introduction of Cre recombinase for marker gene rescue.

Marker rescue is an important molecular technique that enables sequential gene deletions. The Cre-loxP recombination system has been used for marker gene rescue in various organisms, including aspergilli. However, this system requires many time-consuming steps, including construction of a Cre expression plasmid, introduction of the plasmid, and Cre expression in the transformant. To circumvent this laborious process, we investigated a method wherein Cre could be directly introduced into Aspergillus oryzae protoplasts on carrier DNA such as a fragment or plasmid. In this study, we define the carrier DNA (Cre carrier) as a carrier for the Cre enzyme. A mixture of commercial Cre and nucleic acids (e.g., pUG6 plasmid) was introduced into A. oryzae protoplasts using a modified protoplast-polyethylene glycol method, resulting in the deletion of a selectable marker gene flanked by loxP sites. By using this method, we readily constructed a marker gene-rescued strain lacking ligD to optimize homologous recombination. Furthermore, we succeeded in integrative recombination at a loxP site in A. oryzae. Thus, we developed a simple method to use the Cre-loxP recombination system in A. oryzae by direct introduction of Cre into protoplasts using DNA as a carrier for the enzyme.

Increases thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase by fusion of cellulose binding domain derived from Trichoderma reesei.

To improve the thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase (CSLP), the cellulose-binding domain originates from Trichoderma reesei cellobiohydrolase I was engineered into C-terminal region of the CSLP (CSLP-CBD). The CSLP and CSLP-CBD were successfully expressed in the Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from Saccharomyces cerevisiae (α factor). The recombinant CSLP and CSLP-CBD were secreted into culture medium and estimated by SDS-PAGE to be 22 and 27 kDa, respectively. The fusion enzyme was stable at 80 °C and retained more than 80% of its activity after 120-min incubation at this temperature. Our results also found that the fusion of fungal exoglucanase cellulose-binding domain to CSLP is responsible for cellulose-binding capacity. This attribute should make it an attractive applicant for enzyme immobilization.

Characterization and isolation of mutants producing increased amounts of isoamyl acetate derived from hygromycin B-resistant sake yeast.

Hygromycin B is an aminoglycoside antibiotic that inhibits protein synthesis in prokaryotes and eukaryotes. Twenty-four hygromycin B-resistants mutants were isolated from sake yeast, and were divided into three different degrees of strength according to hygromycin B resistance. Three of four hygromycin B strongly resistant mutants produced increased amounts of isoamyl acetate in sake brewing test, although isoamyl alcohol levels remained unchanged. Many hygromycin B-resistants mutants showed higher E/A ratios than K-701 in culture with koji extract medium. Strain HMR-18 produced the largest amount of isoamyl acetate, and its alcohol acetyltransferase (AATFase) activity was 1.3-fold that of K-701. DNA microarray analysis showed that many genes overexpressed in HMR-18 were involved in stress responses (heat shock, low pH, and so on) but HMR-18 showed thermo- and acid-sensitivity. It was strongly resistant to hygromycin B and another aminoglycoside antibiotic, G418.

Phylogenetic and biochemical characterization of the oil-producing yeast Lipomyces starkeyi.

Lipomyces starkeyi is an oleaginous yeast, and has been classified in four distinct groups, i.e., sensu stricto and custers α, ß, and γ. Recently, L. starkeyi clusters α, ß, and γ were recognized independent species, Lipomyces mesembrius, Lipomyces doorenjongii, and Lipomyces kockii, respectively. In this study, we investigated phylogenetic relationships within L. starkeyi, including 18 Japanese wild strains, and its related species, based on internal transcribed spacer sequences and evaluated biochemical characters which reflected the phylogenetic tree. Phylogenetic analysis showed that most of Japanese wild strains formed one clade and this clade is more closely related to L. starkeyi s.s. clade including one Japanese wild strain than other clades. Only three Japanese wild strains were genetically distinct from L. starkeyi. Lipomyces mesembrius and L. doorenjongii shared one clade, while L. kockii was genetically distinct from the other three species. Strains in L. starkeyi s.s. clade converted six sugars, D-glucose, D-xylose, L-arabinose, D-galactose, D-mannose, and D-cellobiose to produce high total lipid yields. The Japanese wild strains in subclades B, C, and D converted D-glucose, D-galactose, and D-mannose to produce high total lipid yields. Lipomyces mesembrius was divided into two subclades. Lipomyces mesembrius CBS 7737 converted D-xylose, L-arabinose, D-galactose, and D-cellobiose, while the other L. mesembrius strains did not. Lipomyces doorenjongii converted all the sugars except D-cellobiose. In comparison to L. starkeyi, L. mesembrius, and L. doorenjongii, L. kockii produced higher total lipid yields from D-glucose, D-galactose, and D-mannose. The type of sugar converted depended on the subclade classification elucidated in this study.

Construction of a new recombinant protein expression system in the basidiomycetous yeast Cryptococcus sp. strain S-2 and enhancement of the production of a cutinase-like enzyme.

Yeast host-vector systems have been very successful in expressing recombinant proteins. However, because there are some proteins that cannot be expressed with existing systems, there is a need for new yeast expression systems. Here we describe a new host-vector system based on the basidiomycetous yeast Cryptococcus sp. strain S-2 (S-2). Two advantages of S-2 are that it naturally produces some very useful enzymes, so it would be a good system for expressing multiple copies of some of its genes, and that, it is a nonhazardous species. The orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) gene (URA5) was selected as a selectable marker for transformation in the new host-vector system. URA5 was isolated and introduced into a uracil auxotroph of S-2 by electroporation. To demonstrate the S-2 system, we selected one of its unique enzymes, a plastic-degrading cutinase-like enzyme (CLE). We were able to insert multiple copies of the CLE gene (CLE1) into the chromosomes in a high fraction of the targeted cells. Under optimal conditions, one transformant exhibited 3.5 times higher CLE activity than the wild type. Expression vectors, including an inducible promoter (the promoter for the xylanase or α-amylase gene), were constructed for recombinant protein production, and green fluorescent protein was expressed under the control of these promoters. The xylanase promoter was more tightly controlled. Furthermore, putting CLE1 under the control of the xylanase promoter, which is induced by xylose, increased CLE activity of the culture medium to approximately 15 times greater than that of the wild type.

Breeding of a new wastewater treatment yeast by genetic engineering.

We previously developed a host vector system for the wastewater treatment yeast Hansenula fabianii J640. The promoter and terminator regions of the gene encoding glucoamylase from H. fabianii J640 were used for a new expression vector, pHFGE-1. The performance of pHFGE-1 was compared with that of the widely used pG-1 transformant vector. H. fabianii J640 (HF-TAMY) cells were transformed with pHFGE-1, and Saccharomyces cerevisiae YPH-499 (SC-TAMY) cells were transformed with pG-1, both of which carried the Taka-amylase. Expression of Taka-amylase by HF-TAMY showed higher than that by SC-TAMY. By using this new system, we bred the new wastewater treatment yeast that shows α-amylase activity. This yeast appears to grow well under experimental wastewater conditions, and is effective in treating model wastewater containing soluble and insoluble starch.

Purification and characterization of a novel aspartic protease from basidiomycetous yeast Cryptococcus sp. S-2.

An aspartic protease (Cap1) was purified from basidiomycetous yeast Cryptococcus sp. S-2 (FERM ABP-10961) using HiTrap DEAE FF column and HiTrap Q HP column chromatography with azocasein as a substrate. Cap1 has a molecular mass of 34 kDa on SDS-PAGE. It was stable up to 50°C with maximum activity at 30°C. Maximum proteolytic activity was observed at pH 5.0. Cap1 was stable in the pH range 3.0-7.0. Its enzyme activity was strongly inhibited by pepstatin A, an inhibitor of aspartic proteases, indicating that Cap1 is an aspartic protease. Cap1 hydrolyzed protein substrates, including BSA, hemoglobin, α-casein, ß-casein, and κ-casein. It showed activity on synthetic substrates, such as MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH2 and MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2. Hydrolysis of the oxidized insulin B chain followed by amino acid sequencing analysis of the cleavage products revealed that 9 of its 30 peptide bonds were hydrolyzed by Cap1. This result was similar to that observed with pig pepsin A and human pepsin A. Cap1 also exhibited milk-clotting activity. We cloned the cDNA of CAP1 gene, which contained a 1254 bp open reading frame encoding a protein of 417 amino acid residues. Homology search in the NCBI database revealed that the amino acid sequence of Cap1 showed less than 39% identity to other known proteins. Therefore, we proposed that Cap1 is a novel aspartic protease.