No abstract

Aspergillus nidulans possessed 16 putative amylolytic genes consisting of 7 alpha-glucosidase (agdA-F), 7 alpha-amylase (amyA-F), and 2 glucoamylase (glaA and B) genes on the genome. Among them, the agdA, agdB, agdE, agdF, amyA, amyB, amyF, and glaB genes were induced by isomaltose. AmyR, a Zn(II)(2)Cys(6) transcription factor, was required for the induction. The isomaltose-inducible genes possessed at least one consensus sequence for AmyR binding, 5'-CGGN(8)CGG, on each promoter region. None of the amylolytic genes was induced by maltose. The mRNA levels of the amylolytic genes except for agdC, amyD, and amyG increased under carbon-starved conditions. Release from CreA-dependent carbon catabolite repression was the main cause of the increase, but, the mRNA levels of agdB, agdF, amyB, amyF, and glaB increased to some extent even in a creA mutant. Therefore, both CreA-dependent and -independent mechanisms are involved in the up-regulation of the amylolytic genes under carbon-starved conditions.

AmyR, a fungal transcriptional activator responsible for induction of amylolytic genes in Aspergillus nidulans, localizes to the nucleus in response to the physiological inducer isomaltose. Maltose, kojibiose, and d-glucose were also found to trigger the nuclear localization of GFP-AmyR. Isomaltose- and kojibiose-triggered nuclear localization was not inhibited by the glucosidase inhibitor, castanospermine, while maltose-triggered localization was inhibited. Thus, maltose itself does not appear to be an direct inducer, but its degraded or transglycosylated product does. Non-metabolizable d-glucose analogues were also able to trigger the nuclear localization, implying that these sugars, except maltose, directly function as the inducers of AmyR nuclear entry. The inducing activity of d-glucose was 4 orders-of-magnitude weaker compared with isomaltose. Although d-glucose has the ability to induce α-amylase production, this activity would generally be masked by CreA-dependent carbon catabolite repression. Significant induction of α-amylase by d-glucose was observed in creA-defective A. nidulans.

Cassava is a starch-containing root crop that is widely used as a raw material in a variety of industrial applications, most recently in the production of fuel ethanol. In the present study, ethanol production from raw (uncooked) cassava flour by simultaneous saccharification and fermentation (SSF) using a preparation consisting of multiple enzyme activities from Aspergillus kawachii FS005 was investigated. The multi-activity preparation was obtained from a novel submerged fermentation broth of A. kawachii FS005 grown on unmilled crude barley as a carbon source. The preparation was found to consist of glucoamylase, acid-stable α-amylase, acid carboxypeptidase, acid protease, cellulase and xylanase activities, and exhibited glucose and free amino nitrogen (FAN) production rates of 37.7 and 118.7 mg/l/h, respectively, during A. kawachii FS005-mediated saccharification of uncooked raw cassava flour. Ethanol production from 18.2% (w/v) dry uncooked solids of raw cassava flour by SSF with the multi-activity enzyme preparation yielded 9.0% (v/v) of ethanol and 92.3% fermentation efficiency. A feasibility study for ethanol production by SSF with a two-step mash using raw cassava flour and the multi-activity enzyme preparation manufactured on-site was verified on a pilot plant scale. The enzyme preparation obtained from the A. kawachii FS005 culture broth exhibited glucose and FAN production rates of 41.1 and 135.5 mg/l/h, respectively. SSF performed in a mash volume of about 1,612 l containing 20.6% (w/v) dry raw cassava solids and 106 l of on-site manufactured A. kawachii FS005 culture broth yielded 10.3% (v/v) ethanol and a fermentation efficiency of 92.7%.