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Author(s): Masayuki Machida (corresponding author) [1]; Kiyoshi Asai [2]; Motoaki Sano [1]; Toshihiro Tanaka [3]; Toshitaka Kumagai [2]; Goro Terai [2, 20]; Ken-Ichi Kusumoto [4]; Toshihide Arima [5]; Osamu Akita [...]

Research has been conducted on Aspergillus parasiticus. The construction of A. parasiticus strain with deleted alfR gene has been carried out in determining the ability of A. sojae alfR coding region to fuse to the inducible amylase gene promoter and the results are reported.

The replacement-type method is used for deleting large chromosomal DNA segments to identify essential and nonessential regions in chromosome, which is the smallest Aspergillus oryzae chromosome and has a large number of nonsyntenic blocks. The results have shown the potential of the method for identifying novel functional genes.

A ku70-deficient strain and a bidirectional marker are used to develop an efficient technique to generate large chromosomal deletions in the koji molds Aspergillus oryzae and A. sojae. The method facilitated deletion of 200-kb and 100-kb sections of A. oryzae and A. sojae, respectively, is applied to the genomic engineering and molecular breeding of filamentous fungi.

The helix-loop-helix (HLH) family of transcriptional factors is a key player in a wide range of developmental processes in organisms from mammals to microbes. We previously identified the bHLH transcription factor SclR in Aspergillus oryzae and found that the loss of SclR function led to significant phenotypic changes, such as rapid protein degradation and cell lysis in dextrin-polypeptone-yeast extract liquid medium. The result implied that SclR is potentially important in both traditional fermentative manufacturing and commercial enzyme production in A. oryzae because of its effect on growth. Therefore, this study presents a comparative assessment at the proteome level of the intracellular differences between an sclR-disrupted strain and a control strain using isobaric tandem mass tag (TMT) labeling for quantification. A total of 5447 proteins were identified, and 568 were differentially expressed proteins (DEPs). Of the DEPs, 251 proteins were increased by 1.5-fold, and 317 proteins were decreased by 1.5-fold in an sclR-disrupted strain compared to the control. The comparison of the quantitative TMT results revealed that SclR was mainly involved in carbon metabolism, especially carbohydrate metabolism. In addition, an enzyme profile by a semi-quantitative method (API-ZYM) indicated that three enzymes (β-galactosidase, α-glucosidase, and α-mannosidase) were significantly less active in the ∆ sclR strain than in the control. Moreover, quantitative RT-PCR showed that the expression of certain genes was changed similarly to their corresponding proteins. These results suggested that a possible function of SclR during growth of A. oryzae is its important involvement in carbohydrate metabolism. [ABSTRACT FROM AUTHOR]

Fungal cellulolytic and hemicellulolytic enzymes are promising tools for industrial hydrolysis of cellulosic biomass; however, the regulatory network underlying their production is not well understood. The recent discovery of the transcriptional activators ClrB and McmA in Aspergillus nidulans implied a novel regulatory mechanism driven by their interaction, experimental evidence for which was obtained from transcriptional and DNA-binding analyses in this study. It was found that ClrB was essential for induced expression of all the genes examined in this study, while McmA dependency of their expression was gene-dependent. DNA-binding studies revealed McmA assisted in the recruitment of ClrB to the cellulose-responsive element (CeRE) in the promoters of eglA and eglB, expression of which was significantly reduced in the mcmA mutant. The CCG triplet within the CeRE served as the recognition sequence for the ClrB monomer. In contrast, ClrB did not require McmA for binding as a homodimer to the CGGN8CCG sequences in the promoter of mndB, expression of which was affected less in the mcmA mutant than in all other examined genes. Thus, there are two types of ClrB-mediated regulation: McmA-assisted and McmA-independent. This novel McmA-ClrB synergistic system provides new insights into the complex regulatory network involved in cellulase and hemicellulase production. [ABSTRACT FROM AUTHOR]

Speradine A is a derivative of cyclopiazonic acid (CPA) found in culture of an Aspergillus tamarii isolate. Heterologous expression of a predicted methyltransferase gene, cpaM, in the cpa biosynthesis gene cluster of A. tamarii resulted in the speradine A production in a 2-oxoCPA producing A. oryzae strain, indicating cpaM is involved in the speradine A biosynthesis. [ABSTRACT FROM AUTHOR]