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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 twodimensional 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 ␣-amylase (TAA) and ␤-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.

To help assess the potential for aflatoxin production by Aspergillus oryzae, the structure of an aflatoxin biosynthesis gene homolog cluster in A. oryzae RIB 40 was analyzed. Although most genes in the corresponding cluster exhibited from 97 to 99% similarity to those of Aspergillus flavus, three genes shared 93% similarity or less. A 257-bp deletion in the aflT region, a frameshift mutation in norA, and a base pair substitution in verA were found in A. oryzae RIB 40. In the aflR promoter, two substitutions were found in one of the three putative AreA binding sites and in the FacB binding site. PCR primers were designed to amplify homologs of aflT, nor-1, aflR, norA, avnA, verB, and vbs and were used to detect these genes in 210 A. oryzae strains. Based on the PCR results, the A. oryzae RIB strains were classified into three groups, although most of them fell into two of the groups. Group 1, in which amplification of all seven genes was confirmed, contained 122 RIB strains (58.1% of examined strains), including RIB 40. Seventy-seven strains (36.7%) belonged to group 2, characterized by having only vbs, verB, and avnA in half of the cluster. Although slight expression of aflR was detected by reverse transcription-PCR in some group 1 strains, including RIB 40, other genes (avnA, vbs, verB, and omtA) related to aflatoxin production were not detected. aflR was not detected in group 2 strains by Southern analysis.Koji molds, Aspergillus oryzae and Aspergillus sojae, have traditionally been used in the brewing industry for the production of sake, miso, and soy sauce. A history of safety (1) and nonproductivity of aflatoxin is well established for industrial strains, and A. oryzae is considered "generally recognized as safe" by the U.S. Food and Drug Administration (41). These fungi belong to the Aspergillus section Flavi, which includes Aspergillus flavus and Aspergillus parasiticus, some of which produce the procarcinogen aflatoxin. It is thought that A. oryzae and A. sojae are taxonomically differentiated from A. flavus and A. parasiticus, respectively (20, 21, 23, 35, 37). It has been demonstrated that most of the 25 identified genes clustered within a specific 70-kb region of the fungal genome are involved in aflatoxin biosynthesis (reviewed in references 3, 13, 22, 40, 45, 49, and 54). Among them, the aflR gene is known to encode a major transcriptional regulator of aflatoxin biosynthesis genes (6,9,19,28,47,55). AflR binds to the consensus sequence 5Ј-TCGN 5 CGR-3Ј (16) found in the promoters of most of the aflatoxin biosynthesis genes (54), including aflR (14, 17). Putative binding sites (14, 17) for the transcription factors AreA (10, 34, 36), PacC (18), and FacB (43) have been identified in the aflR promoter.For A. sojae, several studies (30, 31, 42) have suggested an inability to produce aflatoxin, because mutations have been found in the aflR homolog (46). On the other hand, Kusumoto et al. (25) reported that 39 strains of A. oryzae could be classified into three groups based on fragment analysis by a lo...

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