| Abstract |
We used modified Czapek-Dox (mCD) or dextrin-peptone-yeast extract (DPY) media to cultivate a filamentous fungus, Aspergillus oryzae IAM 2706 by three different cultivation methods, i.e., shaking-flask culture (SFC), agar-plate culture (APC), and membrane-surface liquid culture (MSLC), to identify the differences in cultivation behaviors and gene transcriptional profiles. The fungi cultivated by APC or MSLC secreted a greater number of different proteins/enzymes in larger quantities compared with fungi cultivated by SFC, particularly when DPY medium was used. In particular, the amounts of protease secreted by fungi cultivated via MSLC or APC were much greater compared with SFC. When mCD medium was used, α-amylase activity was barely detectable in all cultures while the activity was detected in MSLC and APC in a quantity that was several times higher than that in SFC using DPY medium. SDS-PAGE analysis and N-terminal amino acid sequences confirmed 6 proteins in the culture supernatants when DPY medium was used. Among these proteins oryzin (an alkaline protease) and α-amylase were detected at much higher levels in APC and MSLC compared with SFC, which was consistent with
the measured activity of the secreted enzymes. However, when mCD medium was used, only oryzin was detected in significant amounts in MSLC and APC. Microarray analyses of the fungi cultivated by SFC, APC or MSLC using either mCD or DPY media indicated that the gene transcriptional profile of the MSLC sample was similar to that of the APC sample but different from that of the SFC sample. When mCD medium was used, most of the genes that were up-regulated 10-folds or greater in the MSLC sample relative to the SFC sample were unknown or predicted proteins. Transcription of the oryzin gene was only slightly up-regulated in the MSLC sample while transcription of the α-amylase gene was slightly down-regulated. On the other hand, when DPY medium was used, many known genes including the oryzin gene were up-regulated in the MSLC sample versus the SFC sample.
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