No abstract

To investigate the structure of the aflatoxin gene cluster in Aspergillus oryzae, 39 strains belonging to this species were examined for the existence of pksA, fas1A, aflR and vbs, and the results compared with those for ver-1 obtained previously. These five genes are involved in aflatoxin biosynthesis in Aspergillus parasiticus. The strains examined were categorized into three groups; group 1, having the five homologs; 2, having ver-1 and vbs; and 3, having vbs homologs. Long-PCR analysis of the regions between the five homologs in A. oryzae IFO 4135, coupled with Southern-hybridization analysis, shows that those homologs are clustered with the same arrangement as in A. parasiticus. These results suggest that directed deletions of the cluster occur in A. oryzae strains. The possible breakpoints of the deletions in the strains of group 2 and 3 were estimated.

In the genome of Aspergillus oryzae, 12 genes have been predicted to encode serine-type carboxypeptidases. However, the carboxypeptidase activities of the proteins encoded by these genes have not yet been confirmed experimentally. In this study, we have constructed three of these 12 genes overexpressing strains using Aspergillus nidulans and characterized their overproduced recombinant proteins. Of these three genes, one was previously named cpI; the other two have not been reported yet, and hence, we named them ocpA and ocpB. The recombinant proteins released amino acid residues from the C terminus of peptides, and the activity of the enzymes was inhibited by phenylmethylsulfonyl fluoride, indicating the enzymes to be serine-type carboxypeptidases. Recombinant OcpA, OcpB, and CpI were stable at 45°C, 55°C, and 55°C, respectively, at a low pH. The enzymatic properties of recombinant OcpB were different from those of any reported serine-type carboxypeptidase. On the other hand, recombinant OcpA had similar enzymatic properties to A. oryzae carboxypeptidases O1 and O2. The DNA and N-terminal amino acid sequences of carboxypeptidases O1 and O2 from A. oryzae IAM2640 were similar to those of OcpA. Result of transcriptional analysis of ocpA, ocpB, and cpI suggest differences in transcriptional regulation between these genes.

A bacterial community analysis, using a culture-independent method (polymerase chain reaction-denaturing gradient gel electrophoresis), detected 17 species of bacteria including species of the genera Tetragenococcus, Lactobacillus, Pediococcus, Weissella Halanaerobium, Clostridium, and Sphingomonas in a traditional salty-fermented fish paste known as pla-ra or pa-daek in Thailand and Laos, which is used as a storage-stable multi-purpose seasoning. The representative genus of lactic acid bacteria seemed to vary in the 10 products collected from Thailand and Laos. Tetragenococci were common in products from central Thailand and Vientiane in Laos which had salinities of not less than 11% and pH values ranging from 5.6 to 6.1. However, lactobacilli were common in products from northern Thailand which had the lowest salinities (8.3–8.6%) and pH values (4.5–4.8) of all the samples examined. Two Lactobacillus and one Tetragenococcus species were detected in one product from northeastern Thailand containing 10% salt. These results suggest that salinity in pla-ra/pa-daek is an important determinant of the representative genus of lactic acid bacteria such as, Tetragenococcus or Lactobacillus. Additionally, differences in the acidity between these two groups seemed to be related to the production of d-/l-lactic acid in the lactic acid bacteria in each product. This is the first study to report a correlation between bacterial community structure and taste components in pla-ra/pa-daek products from various regions. This scientific work on a traditional fermented food will be useful in helping local producers meet differing consumer preferences in various regions.