Kazuhiro Iwashita

Proteomic Analysis of Extracellular Proteins from Aspergillus oryzae Grown under Submerged and Solid-State Culture Conditions

ABSTRACT Filamentous fungi are widely used for the production of homologous and heterologous proteins. Recently, there has been increasing interest in Aspergillus oryzae because of its ability to produce heterologous proteins in solid-state culture. To provide an overview of protein secretion by A. oryzae in solid-state culture, we carried out a comparative proteome analysis of extracellular proteins in solid-state and submerged (liquid) cultures. Extracellular proteins prepared from both cultures sequentially from 0 to 40 h were subjected to two-dimensional electrophoresis, and protein spots at 40 h were identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. We also attempted to identify cell wall-bound proteins of the submerged culture. We analyzed 85 spots from the solid-state culture and 110 spots from the submerged culture. We identified a total of 29 proteins, which were classified into 4 groups. Group 1 consisted of extracellular proteins specifically produced in the solid-state growth condition, such as glucoamylase B and alanyl dipeptidyl peptidase. Group 2 consisted of extracellular proteins specifically produced in the submerged condition, such as glucoamylase A (GlaA) and xylanase G2 (XynG2). Group 3 consisted of proteins produced in both conditions, such as xylanase G1. Group 4 consisted of proteins that were secreted to the medium in the solid-state growth condition but trapped in the cell wall in the submerged condition, such as α-amylase (TAA) and β-glucosidase (Bgl). A Northern analysis of seven genes from the four groups suggested that the secretion of TAA and Bgl was regulated by trapping these proteins in the cell wall in submerged culture and that secretion of GlaA and XynG2 was regulated at the posttranscriptional level in the solid-state culture.

Whole-Genome Sequencing of Sake Yeast Saccharomyces cerevisiae Kyokai no. 7

The term ‘sake yeast’ is generally used to indicate the Saccharomyces cerevisiae strains that possess characteristics distinct from others including the laboratory strain S288C and are well suited for sake brewery. Here, we report the draft whole-genome shotgun sequence of a commonly used diploid sake yeast strain, Kyokai no. 7 (K7). The assembled sequence of K7 was nearly identical to that of the S288C, except for several subtelomeric polymorphisms and two large inversions in K7. A survey of heterozygous bases between the homologous chromosomes revealed the presence of mosaic-like uneven distribution of heterozygosity in K7. The distribution patterns appeared to have resulted from repeated losses of heterozygosity in the ancestral lineage of K7. Analysis of genes revealed the presence of both K7-acquired and K7-lost genes, in addition to numerous others with segmentations and terminal discrepancies in comparison with those of S288C. The distribution of Ty element also largely differed in the two strains. Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty elements in K7. Sequence comparisons suggest that these gene conversions were caused by cDNA-mediated recombination of Ty elements. The present study advances our understanding of the functional and evolutionary genomics of the sake yeast.

Recent studies of protein secretion by filamentous fungi.

Filamentous fungi have been widely exploited for the homologous and heterologous protein production, because of the high capacity of their protein secretion machinery. However, the production of heterologous proteins is often limited while the production of homologous proteins can be very high. Various researches have reported the methods for overcoming this problem and some techniques, such as the fusion gene system, improve the production of heterologous proteins. Recently, the molecular biological study of solid-state culture attracts the attention, because the long history of biological studies has shown that the productivity of protein in the solid-state culture frequently exceeds the productivity of protein in the submerged culture. The recent researches of solid-state culture have revealed the new aspects of protein production in filamentous fungi. Solid-state specific gene expression was observed in the glaB and pepA genes of Aspergillus oryzae. A GC-box and HSE element of the glaB promoter region affected solid-state specific gene expression of this gene. Solid-state culture-specific release of enzymes from the cell wall was also observed in the production of beta-glucosidases in Aspergillus kawachii. Extracellular soluble polysaccharide (ESP) from A. kawachii was concerned with the location of beta-glucosidases. Moreover, ESP and the cell wall fraction of A. kawachii were shown to be involved in the stability of beta-glucosidases. The knowledge of the molecular biology of solid-state culture should provide new approaches for the production of both homologous and heterologous proteins in both submerged culture and solid-state culture of filamentous fungi.

Effects of Culture Conditions on Ergosterol Biosynthesis by Saccharomyces cerevisiae

Ergosterol is an essential component of yeast cells that maintains the integrity of the membrane. It was investigated as an important factor in the ethanol tolerance of yeast cells. We investigated the effects of brewing conditions on the ergosterol contents of S. cerevisiae K-9, sake yeast, several kinds of Saccharomyces cerevisiae that produce more than 20% ethanol, and X2180-1A, laboratory yeast. K-9 had a higher total ergosterol contents under all the conditions we examined than X2180-1A. Ethanol and hypoxia were found to have negative and synergistic effects on the total ergosterol contents of both strains, and significantly reduced the free ergosterol contents of X2180-1A but only slightly reduced those of K-9. The maintenance of free ergosterol contents under brewing conditions might be an important character of sake yeast strains. DNA microarray analysis also showed higher expression of ergosterol biosynthesis genes in K-9 than in X2180-1A.

Aspergillus oryzae atfA controls conidial germination and stress tolerance.

We compared atfA and atfB, the genes encoding the respective ATF/CREB-type transcription factors in Aspergillus oryzae. The germination ratio of DeltaatfA conidia was low without any stress, unlike that of DeltaatfB conidia. The DeltaatfA conidia were more sensitive to oxidative stress than the DeltaatfB conidia, which are also sensitive to oxidative stress. We compared the gene expressions of these strains by using a DNA microarray, GeneChip. Almost all the genes regulated by atfB were also regulated by atfA, but atfA also regulated many genes that were not regulated by atfB, including some genes putatively involved in oxidative stress resistance. The level of glutamate, the major amino acid in A. oryzae conidia, was significantly low only in the DeltaatfA conidia, and the glycerol accumulation during germination was not observed only in the DeltaatfA strain. We therefore concluded that atfA is involved in germination via carbon and nitrogen source metabolism.

Aspergillus oryzae atfB encodes a transcription factor required for stress tolerance in conidia.

Using an Aspergillus oryzae EST database, we identified a gene encoding a transcription factor (atfB), which is a member of the ATF/CREB family. Expression of atfB was barely detectable during vegetative growth, but was readily detected during conidiation in solid-state culture. Microarray analyses showed that expression of many other genes, including catalase (catA), were downregulated in an atfB-disruptant. The expression of most of these genes was upregulated in the wild-type strain during the conidiation phase in solid-state culture, and the expression pattern was similar to that of atfB itself. In the absence of stress, e.g. heat-shock or hydrogen peroxide, the conidial germination ratios for the DeltaatfB strain and the wild-type strain were similar, but the stress tolerance of conidia carrying the DeltaatfB deletion was less than that of the wild-type conidia. CRE-like DNA motifs, which are bound by ATF/CREB proteins, were found in the promoters of most of the downregulated genes in the DeltaatfB strain. Thus, atfB appears to encode a transcription factor required for stress tolerance in conidia.

A new method for isolation of S -adenosylmethionine (SAM)-accumulating yeast

S-Adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms, and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression, liver disease, and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM. Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study. In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin, which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolated through this method. These mutants accumulated 1.7–5.5 times more SAM than their parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene, and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating SAM.

Aflatoxin non-productivity of Aspergillus oryzae caused by loss of function in the aflJ gene product.

Aspergillus oryzae, although closely related to Aspergillus flavus, does not produce aflatoxin (AF). A. oryzae RIB strains can be classified into three groups (group 1-3) based on the structure of the AF biosynthesis gene homolog cluster (AFHC). In group 1 strains, where AFHC is present, the expression level of the aflR gene is extremely low and there is no expression of the other four AF homologue genes (avnA, verB, omtA and vbs). We conducted a detailed structural comparison of AFLR ORF and AFLJ ORF from A. oryzae and A. flavus and identified several amino-acid substitutions. If these substitutions induce inactivation of AFLR and AFLJ, AF biosynthesis of A. oryzae will be doubly inhibited at the transcriptional and translational level. In this study, we transferred aflR and aflJ to A. oryzae RIB67, a group 2 strain where more than half of AFHC is missing. Under control of the pgkA promoter, aflR and aflJ was expressed and avnA, verB, omtA and vbs gene expression were monitored by RT-PCR. We prepared six types of forced-expression vectors, including aflR (from A. oryzae RIB40 or its three mutants) or aflJ (from A. oryzae RIB40 or A. flavus RIB4011). RT-PCR analysis showed that transformants containing aflJ from A. oryzae displayed no expression of AF biosynthetic homologue genes, whereas aflR substitutions had no such effect. These results strongly suggest that the amino-acid substitutions in AFLJ of A. oryzae induce inactivation at the protein level.

Treatment and phosphorus removal from high-concentration organic wastewater by the yeast Hansenula anomala J224 PAWA.

A flocculent yeast, Hansenula anomala J224 PAWA, bred in this study, accumulated twice as much phosphorus as the wild type. Over a 30-d period, PAWA removed 70-80% of dissolved total phosphorus from sweet-potato and barley shochu wastewaters (alcoholic distillery wastewaters) while the wild type removed only 30%. Waste sludge was easily separated from effluent wastewater because PAWA cells made large flocks that rapidly settled. Component analysis suggested that PAWA sludge could be used as a protein source for feedstuff and as a phosphorus source for fertilizer. Under anaerobic conditions, denitrification was rapid, resulting in the removal of large amounts of nitrogen from barley shochu wastewater. These results suggest that small shochu manufacturers could benefit from using PAWA to remove phosphorus and organic compounds and then by using a combination of the upflow anaerobic sludge blanket and the downflow hanging sponge method (UASB-DHS method) for nitrification/denitrification.

Breeding of wastewater treatment yeasts that accumulate high concentrations of phosphorus

Inorganic phosphate is an essential nutrient. In general, microorganisms take up phosphorus when the extracellular phosphorus concentration is low, but not when it is high. In Saccharomyces cerevisiae, the major phosphate transporters, such as Pho84p, and acid phosphatases (APases), such as Pho5p, are regulated in parallel by the phosphate signal transduction pathway (PHO pathway). We found that PHO mutants expressing PHO84 and PHO5, even under high-P conditions, could take up phosphorus at twice the rate of the wild-type strain. The regulatory pathway for phosphorus accumulation in two wastewater treatment yeasts, Hansenula fabianii J640 and Hansenula anomala J224-1, was found to be similar to that in S. cerevisiae. We screened for mutants of these yeasts that constitutively expressed APase. Such mutants formed blue colonies on high phosphorus concentration agar plates containing 5-bromo-4-chloro-3-indolylphosphate (X-phosphate). We found four mutants of H. fabianii J640 and one mutant of H. anomala J224-1 that accumulated from 2.2 to 3.5 times more phosphorus than the parent strains. The growth rates and abilities to remove dissolved total nitrogen and dissolved organic carbon of the mutants were similar to those of the parent strains. In addition, the mutants removed 95% of dissolved total phosphorus from shochu wastewater, while the parent strain removed only 50%.