Isolation and analysis of a sake yeast mutant with phenylalanine accumulation.

Sake is a traditional Japanese alcoholic beverage brewed by the yeast Saccharomyces cerevisiae. Since the consumption and connoisseurship of sake has spread around the world, the development of new sake yeast strains to meet the demand for unique sakes has been promoted. Phenylalanine is an essential amino acid that is used to produce proteins and important signaling molecules involved in feelings of pleasure. In addition, phenylalanine is a precursor of 2-phenylethanol, a high-value aromatic alcohol with a rose-like flavor. As such, adjusting the quantitative balance between phenylalanine and 2-phenylethanol may introduce value-added qualities to sake. Here, we isolated a sake yeast mutant (strain K9-F39) with phenylalanine accumulation and found a missense mutation on the ARO80 gene encoding the His309Gln variant of the transcriptional activator Aro80p involved in the biosynthesis of 2-phenylethanol from phenylalanine. We speculated that mutation of ARO80 would decrease transcriptional activity and suppress the phenylalanine catabolism, resulting in an increase of intracellular phenylalanine. Indeed, sake brewed with strain K9-F39 contained 60% increase in phenylalanine, but only 10% less 2-phenylethanol than sake brewed with the parent strain. Use of the ARO80 mutant in sake brewing may be promising for the production of distinctive new sake varieties. ONE-SENTENCE SUMMARY: The ARO80 mutant is appropriate for controlling the content of phenylalanine and 2-phenylethanol.

Pyruvate metabolism redirection for biological production of commodity chemicals in aerobic fungus Aspergillus oryzae.

Pyruvate is a central metabolite for the biological production of various chemicals. In eukaryotes, pyruvate produced by glycolysis is used in conversion to ethanol and lactate and in anabolic metabolism in the cytosol, or is transported into the mitochondria for use as a substrate in the tricarboxylic acid (TCA) cycle. In this study, we focused on controlling pyruvate metabolism in aerobic microorganisms for the biological production of various chemicals. We successfully improved productivity by redirecting pyruvate metabolism in the aerobic filamentous fungus Aspergillus oryzae via the deletion of two genes that encode pyruvate decarboxylase and mitochondrial pyruvate carriers. Production of ethanol as a major byproduct was completely inhibited, and the limited translocation of pyruvate into the mitochondria shifted the metabolism from respiration for energy conversion to the effective production of lactate or 2,3-butandiole, even under aerobic conditions. Metabolomic and transcriptomic analyses showed an emphasis on glycolysis and a repressed TCA cycle. Although the dry mycelial weights of the deletion mutants were reduced compared with those of wild type, the titer and yields of the target products were drastically increased. In particular, the redirection of pyruvate metabolism shifted from anabolism for biomass production to catabolism for the production of target chemicals. Conclusively, our results indicate that the redirection of pyruvate metabolism is a useful strategy in the metabolic engineering of aerobic microorganisms.

Effects of mutations of GID protein-coding genes on malate production and enzyme expression profiles in Saccharomyces cerevisiae.

During alcohol fermentation, Saccharomyces cerevisiae produces organic acids, including succinate, acetate, and malate. Since malate contributes to the pleasant flavor of sake (a Japanese alcoholic beverage), various methods for breeding high-malate-producing yeast have been developed. We previously isolated a high-malate-producing strain and found that a missense mutation in GID4 was responsible for the high-malate-producing phenotype. Gid4 is a component of the GID (glucose-induced degradation-deficient) complex and stimulates the catabolic degradation of gluconeogenic enzymes. In this study, the mechanism by which this mutation led to high malate production in yeast cells was investigated. The evaluation of disruptants and mutants of gluconeogenic enzymes revealed that cytosolic malate dehydrogenase (Mdh2) participated in the malate production. Furthermore, target proteome analysis indicated that an increase in malate production resulted from the accumulation of Mdh2 in gid4 disruptant due to the loss of GID complex-mediated degradation. Next, we investigated the effects of GID protein-coding genes (GID1-GID9) on organic acid production and enzyme expression profiles in yeast. The disruptants of GID1, 2, 3, 4, 5, 8, and 9 exhibited high malate production. Comparison of protein abundance among the GID disruptants revealed variations in protein expression profiles, including in glycolysis and tricarboxylic acid cycle-related enzymes. The high-malate-producing disruptants showed the activation of several glycolytic enzymes and a reduction in enzymes involved in the conversion of pyruvate to ethanol. Our results suggest that high-malate-producing disruptants adapt their metabolism to produce malate in excess via the regulation of protein expression in glucose assimilation and ethanol fermentation. KEY POINTS: An increase in malate level of GID4 mutant resulted from the accumulation of Mdh2. The disruptants of GID1, 2, 3, 4, 5, 8, and 9 showed high malate production. The protein expression profiles in the GID disruptants differed from one another.

Effects of a novel variant of the yeast γ-glutamyl kinase Pro1 on its enzymatic activity and sake brewing.

Sake is a traditional Japanese alcoholic beverage brewed with the yeast Saccharomyces cerevisiae. Sake taste is affected by sugars, organic acids, and amino acids. We previously isolated mutants resistant to the proline analogue azetidine-2-carboxylate derived from a diploid sake yeast strain. Some of the mutants produced a greater amount of proline in the brewed sake. One of them (strain K-9-AZC) carried a novel mutation in the PRO1 gene encoding the Gln79His variant of the γ-glutamyl kinase Pro1, a key enzyme in proline biosynthesis in S. cerevisiae. This mutation resulted in extreme desensitization to feedback inhibition by proline, leading to proline overproduction. Interestingly, sake brewed with K-9-AZC contained 3.7-fold more proline, but only 25% less succinate than sake brewed with the parent strain. Metabolome analysis suggests that the decrease in succinate was attributable to a lower level of 2-oxoglutarate, which is converted into glutamate. The approach here could be a practical method for breeding of yeast strains involved in the diversity of sake taste.

Sake lees extract improves hepatic lipid accumulation in high fat diet-fed mice.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is increasing worldwide as one of the leading causes of chronic liver disease. Sake lees (SL) are secondary products of sake manufacturing and are considered to have beneficial effects on human health. To investigate these effects, we used high fat diet (HFD)-fed mice treated with or without the SL extract. METHOD: Mice were the HFD ad libitum for 8 weeks and were administered 500 µL of distilled water with or without the SL extract (350 mg/mL) by a feeding needle daily for the last 4 weeks. Food intake, body weight, and liver weight were measured. Triacylglycerol content and the mRNA and protein expression levels of various lipid and glucose metabolism-related genes were determined in liver tissues. The levels of triglyceride, free fatty acids, glucose, insulin, and liver cell damage markers were determined in serum. Fatty acid-induced lipid accumulation in HepG2 cells was assessed in the presence or absence of the SL extract. RESULTS: Mice fed a HFD and treated with the SL extract demonstrated a significant reduction in hepatic lipid accumulation and mRNA and protein levels of peroxidome proliferator-activated receptor γ (PPARγ), PPARα, CD36, and phosphoenolpyruvate carboxykinase 1 in the liver, while the SL extract did not affect body weight and food intake. Moreover, insulin resistance and hepatic inflammation in HFD-fed mice improved after administration of the SL extract. In HepG2 cells, the SL extract suppressed fatty acid-induced intracellular lipid accumulation. CONCLUSIONS: These findings suggest that treatment with the SL extract could potentially reduce the risk of NAFLD development, and that the SL extract may be clinically useful for the treatment of NAFLD.

Production of the natural iron chelator deferriferrichrysin from Aspergillus oryzae and evaluation as a novel food-grade antioxidant.

BACKGROUND: Deferriferrichrysin (Dfcy) is a siderophore found in foods fermented by Aspergillus oryzae and is a promising candidate for an antioxidant food additive because of its high binding constant toward iron. However, the Dfcy concentration is typically low in foods and cultures. RESULTS: We optimised culture conditions to improve Dfcy production to 2800 mg L(-1) from 22.5 mg L(-1) under typical conditions. Then, we evaluated the potential of Dfcy as a food additive by measuring its safety, stability, and antioxidant activity. Dfcy was sufficiently stable that over 90% remained after pasteurisation at 63 °C for 30 min at pH 3-11, or after sterilisation at 120 °C for 4 min at pH 4-6. Dfcy showed high antioxidant activity in an oil-in-water model, where inhibition of lipid oxidation was measured by peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) assays. Dfcy decreased PV and TBARS by 83% and 75%, respectively. Antioxidant activity of Dfcy was equal to or higher than that of the synthetic chelator EDTA. CONCLUSION: Our study provides the first practical method for production of Dfcy. Dfcy can be a novel food-grade antioxidant and the first natural alternative to the synthesised iron chelator EDTA. © 2015 Society of Chemical Industry.

Effective saccharification of kraft pulp by using a cellulase cocktail prepared from genetically engineered Aspergillus oryzae.

Kraft pulp is a promising feedstock for bioproduction. The efficiency of kraft pulp saccharification was improved by using a cellulase cocktail prepared from genetically engineered Aspergillus oryzae. Application of the cellulase cocktail was demonstrated by simultaneous saccharification and fermentation, using kraft pulp and non-cellulolytic yeast. Such application would make possible to do an efficient production of other chemicals from kraft pulp.

Aspergillus oryzae-based cell factory for direct kojic acid production from cellulose.

BACKGROUND: Kojic acid (5-Hydroxy-2-(hydroxymethyl)-4-pyrone) is one of the major secondary metabolites in Aspergillus oryzae. It is widely used in food, pharmaceuticals, and cosmetics. The production cost, however, is too high for its use in many applications. Thus, an efficient and cost-effective kojic acid production process would be valuable. However, little is known about the complete set of genes for kojic acid production. Currently, kojic acid is produced from glucose. The efficient production of kojic acid using cellulose as an inexpensive substrate would help establish cost-effective kojic acid production. RESULTS: A kojic acid transcription factor gene over-expressing the A. oryzae strain was constructed. Three genes related to kojic acid production in this strain were transcribed in higher amounts than those found in the wild-type strain. This strain produced 26.4 g/L kojic acid from 80 g/L glucose. Furthermore, this strain was transformed with plasmid harboring 3 cellulase genes. The resultant A. oryzae strain successfully produced 0.18 g/L of kojic acid in 6 days of fermentation from the phosphoric acid swollen cellulose. CONCLUSIONS: Kojic acid was produced directly from cellulose material using genetically engineered A. oryzae. Because A. oryzae has efficient protein secretion ability and secondary metabolite productivity, an A. oryzae-based cell factory could be a platform for the production of various kinds of bio-based chemicals.

Structural analysis of cerebrosides from Aspergillus fungi: the existence of galactosylceramide in A. oryzae.

Glucosylceramide and galactosylceramide were detected in three Aspergillus species: Aspergillus oryzae, Aspergillus sojae and Aspergillus. awamori, using borate-coated TLC. The cerebrosides from A. oryzae were further purified by ion exchange and iatrobeads column chromatographies with or without borate, and determined the composition of sugar, fatty acid and sphingoid base by GC/MS, MALDI-TOF/MS and (1)H-NMR. We identified them as ß-glucosylceramide and ß-galactosylceramide. The ceramide moiety of both cerebrosides consisted mainly of 2-hydroxystearic acid and either 9-methyl-octadeca-4, 8-sphingadienine or octadeca-4, 8-sphingadienine. To our knowledge, this is the first study to provide evidence for the presence of ß-galactosylceramide in A. oryzae.

Effect of yeast chromosome II aneuploidy on malate production in sake brewing.

Chromosome aneuploidy is a common phenomenon in industrial yeast. Aneuploidy is considered one of the strategies to enhance the industrial properties of Saccharomyces cerevisiae strains. However, the effects of chromosomal aneuploidy on the brewing properties of sake have not been extensively studied. In this study, sake brewing was performed using a series of genome-wide segmental duplicated laboratory S. cerevisiae strains, and the effects of each segmentally duplicated region on sake brewing were investigated. We found that the duplication of specific chromosomal regions affected the production of organic acids and aromatic compounds in sake brewing. As organic acids significantly influence the taste of sake, we focused on the segmental duplication of chromosome II that alters malate levels. Sake yeast Kyokai No. 901 strains with segmental chromosome II duplication were constructed using a polymerase chain reaction-mediated chromosomal duplication method, and sake was brewed using the resultant aneuploid sake yeast strains. The results showed the possibility of developing sake yeast strains exhibiting low malate production without affecting ethanol production capacity. Our study revealed that aneuploidy in yeast alters the brewing properties; in particular, the aneuploidy of chromosome II alters malate production in sake brewing. In conclusion, aneuploidization can be a novel and useful tool to breed sake yeast strains with improved traits, possessing industrial significance.

Activation mechanism of melB tyrosinase from Aspergillus oryzae by acidic treatment.

The pro form of recombinant tyrosinase from Aspergillus oryzae (melB) shows no catalytic activity, but acid treatment (around pH 3.5) of protyrosinase activates it to induce tyrosinase activity. Circular dichroism spectra, gel filtration analysis, and colorimetric assay have indicated that acid treatment around pH 3.5 induced the disruption of the conformation of the C-terminal domain covering the enzyme active site. These structural changes induced by the acid treatment may open the entrance to the enzyme active site for substrate incorporation. To compare the mechanism of hydroxylation by the acid-treated tyrosinase with that by trypsin-treated tyrosinase, a detailed steady-state kinetic analysis of the phenolase activity was performed by monitoring the O(2)-consumption rate using a Clark-type oxygen electrode. The results clearly show that the phenolase activity (phenol hydroxylation) of the activated tyrosinase involves an electrophilic aromatic substitution mechanism as in the case of mushroom tyrosinase (Yamazaki and Itoh in J. Am. Chem. Soc. 125:13034-13035, 2003) and activated hemocyanin with urea (Morioka et al. in J. Am. Chem. Soc. 128:6788-6789, 2006).

Synthesis and functional analysis of deferriferrichrysin derivatives: application to colorimetric pH indicators.

Deferriferrichrysin belongs to the siderophore peptide family which are Fe(III)-coordinating cyclic peptides. The common structure of this family is three consecutive hydroxamate moieties, such as N(δ)-acetyl-N(δ)-hydroxy-l-ornithine (Aho). We have designed two deferriferrichrysin derivatives where three Aho residues were arranged as: cyclo(-Aho-Gly-Aho-Gly-Aho-Gly-) and cyclo(-Aho-Ser-Aho-Ser-Aho-Ser-). Comparative evaluation of the physicochemical properties of their Fe(III) complexes revealed that naturally occurring deferriferrichrysin formed a more stable Fe(III) complex when compared with the two derivatives. This result shows that three consecutive Aho residues are indispensable for high affinity Fe(III) binding by deferriferrichrysin. Of note, the observed pH-dependent chromogenic response of the Fe(III) complexes of the derivatives suggests that these two derivatives should function as sensitive pH indicators in acidic environments.

High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.

Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.

Identification of regulatory elements in the glucoamylase-encoding gene (glaB) promoter from Aspergillus oryzae.

The Aspergillus oryzae glucoamylase-encoding gene glaB is expressed specifically and strongly only during solid-state cultivation (SSC). To elucidate the basis for the specificity, the glaB promoter was analyzed by electrophoretic gel mobility shift assay (EMSA) which indicated two protein-binding elements from -382 to -353 and from -332 to -313. To confirm that these regions contained cis-elements, deletion analysis of the promoter was undertaken using ß-glucuronidase as a reporter. The results of the deletion analysis were consistent with the EMSA results. The promoter missing the -332 to -313 element was not induced by low water activity stress during SSC.

Multifunctions of MelB, a fungal tyrosinase from Aspergillus oryzae.

The pro form of melB tyrosinase from the melB gene of Aspergillus oryzae was over-produced from E. coli and formed a homodimer that exhibited the spectral features of met-tyrosinase. In the presence of NH(2)OH (reductant), the proenzyme bound dioxygen to give a stable (µ-η(2):η(2) -peroxo)dicopper(II) species (oxy form), thus indicating that the pro form tyrosinase can function as an oxygen carrier or storage protein like hemocyanin. The pro form tyrosinase itself showed no catalytic activity toward external substrates, but proteolytic digestion with trypsin activated it to induce tyrosinase activity. Mass spectroscopy analyses, mutagenesis experiments, and colorimetry assays have demonstrated that the tryptic digestion induced cleavage of the C-terminal domain (Glu458-Ala616), although the dimeric structure of the enzyme was retained. The structural changes induced by proteolytic digestion might open the entrance to the enzyme active site for substrate incorporation.

Synthesis and application of an Nδ-acetyl-Nδ-hydroxyornithine analog: identification of novel metal complexes of deferriferrichrysin.

Synthesis of Fmoc-protected N(δ)-acetyl-N(δ)-(tert-butoxy)-l-ornithine has revealed it to be a metal-chelating amino-acid precursor. This protected amino acid was compatible with the preparation of ferrichrome peptides by standard Fmoc-based solid-phase peptide synthesis. Evaluation of deferriferrichrysin for metal ion chelation revealed that zirconium(IV) and titanium(IV) formed complexes with deferriferrichrysin.

Aspergillus oryzae FaeA is responsible for the release of ferulic acid, a precursor of off-odor 4-vinylguaiacol in sake brewing.

4-Vinylguaiacol (4-VG) is one of the most common off-flavors found in sake. 4-VG is produced from its precursor, ferulic acid, which is a component of the cell wall of the rice endosperm. The release of ferulic acid in sake brewing is thought to be mediated by feruloyl esterase produced by either Aspergillus oryzae or Saccharomyces cerevisiae. To investigate the effect of FaeA, a feruloyl esterase produced by A. oryzae, its loss-of-function strain was produced by genome co-editing. The feruloyl esterase activity of the faeA-deficient strain was drastically reduced. Sake was fermented using koji with S. cerevisiae strain G046, which can convert ferulic acid to 4-VG. Fermented sake was analyzed by measuring the 4-VG content and sensory evaluation. 4-VG content was reduced to approximately 10% of that of sake fermented with control koji. Sensory evaluation revealed that 4-VG was almost undetectable. Our findings showed that disruption of faeA in A. oryzae is a promising strategy to reduce 4-VG off-flavors in sake.

Post-translational His-Cys cross-linkage formation in tyrosinase induced by copper(II)-peroxo species.

Autocatalytic formation of His-Cys cross-linkage in the enzyme active site of tyrosinase from Aspergillus oryzae has been demonstrated to proceed by the treatment of apoenzyme with Cu(II) under aerobic conditions, where a (µ-η(2):η(2)-peroxo)dicopper(II) species has been suggested to be involved as a key reactive intermediate.

Isolation of a novel promoter for efficient protein expression by Aspergillus oryzae in solid-state culture.

A novel promoter from a hemolysin-like protein encoding the gene, hlyA, was characterized for protein overexpression in Aspergillus oryzae grown in solid-state culture. Using endo-1,4-ß-glucanase from A. oryzae (CelA) as the reporter, promoter activity was found to be higher than that of the α-amylase (amyA) and manganese superoxide dismutase (sodM) genes not only in wheat bran solid-state culture but also in liquid culture. Expression of the A. oryzae endoglucanase CelB and two heterologous endoglucanases (TrEglI and TrEglIII from Trichoderma reesei) under the control of the hlyA promoter were also found to be stronger than under the control of the amyA promoter in A. oryzae grown in wheat bran solid-state culture, suggesting that the hlyA promoter may be useful for the overproduction of other proteins as well. In wheat bran solid-state culture, the productivity of the hlyA promoter in terms of protein produced was high when the cultivation temperature was 30°C or 37°C, when the water content was 0.6 or 0.8 ml/g wheat bran, and from 48 to 72 h after inoculation. Because A. oryzae sporulated actively under these conditions and because hemolysin has been reported to play a role in fungal fruiting body formation, high-level expression of hlyA may be related to sporulation.

Display of both N- and C-terminal target fusion proteins on the Aspergillus oryzae cell surface using a chitin-binding module.

A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein.