Establishment of a hyper-protein production system in submerged Aspergillus oryzae culture under tyrosinase-encoding gene (melO) promoter control

UV-mediated mutagenesis generated a high glucoamylase-producing mutant of Aspergillus oryzae exhibiting strong melanization in solid-state culture. Expression of the glucoamylase-encoding gene (glaB), which is specifically expressed in solid-state culture, and the tyrosinase-encoding gene (melO), was analyzed using an E. coli beta-glucuronidase (GUS) reporter assay to investigate this phenomenon. Although no common regulation was found for melO and glaB expression, the former was greatly enhanced in submerged culture. Interestingly, the melO promoter was about four times stronger for GUS production than the powerful promoters amyB, glaA, and modified agdA, previously isolated for industrial heterologous gene expression in A. oryzae. These findings indicated that the melO promoter would be suitable for hyper-production of heterologous protein in Aspergillus. The glaB-type glucoamylase selected as the target protein was produced in a submerged culture of A. oryzae under the control of the melO promoter. The maximum yield was 0.8 g/l broth, and the total extracellular protein purity was 99%. Repeated batch culture, to improve productivity, gave a maximum yield of 3.3 g/l broth. The importance of this work is in the establishment of a both high-level and high-purity protein overproduction system in A. oryzae by use of the melO promoter.     

Improvement of the glaB promoter expressed in solid-state fermentation (SSF) of Aspergillus oryzae

The glucoamylase-encoding gene (glaB) promoter should be very useful for recombinant protein production in solid-state fermentation (SSF) of Aspergillus oryzae. A 97-bp fragment containing the cis-element of the glaB promoter was inserted into the glaA promoter, which was little expressed in SSF. The chimeric promoter showed about a 24-fold increase in promoter activity in SSF. Eight copies of the 97-bp fragment were tandemly fused with the glaB promoter. The improved promoter showed about a 4.6-fold increase in promoter activity in SSF. The glaB gene was overexpressed under control of the improved glaB promoter in SSF. Recombinant glucoamylase production reached about 1524 mg/kg-broth for 2 d. The improved glaB promoter should be very useful for overproduction of a recombinant protein in SSF of A. oryzae.     

Contribution Ratios of amyA, amyB, amyC Genes to High-Level α-Amylase Expression in Aspergillus oryzae

Aspergillus oryzae strains express α-amylases abundantly, and the genome reference strain RIB40 has three α-amylase genes (amyA, amyB, and amyC). However, there is no information on the contribution ratios of individual α-amylase genes to total expression. In this study, we generated single, double, and triple disruptants of α-amylase genes by employing a strain (ΔligD) with high gene-targeting efficiency and pyrG marker recycling in A. oryzae. All the disruptants showed reduced activities of α-amylases, and the triple disruptant completely lost activity. Comparative analyses of the activities and mRNA amounts of the α-amylases suggest that the contribution of amyA to the α-amylase expression is smaller than those of amyB and amyC. The present study suggests that the ability to express a large amount of α-amylases in A. oryzae is attributed to gene duplication of genes such as amyB and amyC.     

Heat-stress-dependency and developmental modulation of gene expression: the potential of house-keeping genes as internal standards in mRNA expression profiling using real-time RT-PCR

The potential of different house-keeping genes for their use as internal standards of gene expression under changing environmental conditions and in different organs of plants was assessed. Using real-time PCR mRNA levels were precisely quantified for preselected actin and ribosomal protein genes in Arabidopsis thaliana (L.) Heinh. and Nicotiana tabacum L. grown at normal temperature and following heat stress. In tobacco leaves the mRNA levels of the constitutively expressed ribosomal protein gene Nt-L25 and the actin genes Nt-ACT9 and At-ACT66 were strongly reduced (to approximately 10%) during heat stress. Heat stress applied at the temperature optimum (37 degrees C) for elicitation of a heat stress response to Arabidopsis leaves resulted in a strong induction (several thousand-fold) of the mRNA heat shock protein genes, At-HSP17.6 and At-HSP18.2. Concomitantly, the mRNA levels of constitutively expressed actin 2 (At-ACT2) and ribosomal protein L23 (At-L23a) genes were reduced to approximately 50% of the levels in leaves incubated at room temperature. Conversely, under severe heat stress conditions (44 degrees C), the induction of At-HSP17.6 and At-HSP18.2 mRNAs was insignificant, the mRNA levels of At-ACT2 remained at approximately the same levels as in leaves incubated at room temperature, whereas the mRNA level of At-L23 declined. The mRNA levels of At-ACT2 and At-L23a examined in stem, flower and siliques of Arabidopsis plants grown under non-stress condition showed differential alterations; the mRNA level of ribosomal protein L23 correlates with the metabolic activity of tissues. The potential use of house-keeping gene expression as standards in expression profiling and the mechanisms modulating the mRNA levels are discussed.     

Differential display of skin mRNAs regulated under varying environmental conditions in a mudskipper

To understand the molecular mechanisms underlying the terrestrial adaptation, as well as adaptation to different salinities, of the euryhaline and amphibious mudskipper ( Periophthalmus modestus), we have looked for the skin mRNAs that change during varying environmental conditions. Using differential mRNA display polymerase chain reaction, we compared skin mRNAs in mudskipper transferred from isotonic 30% seawater to fresh water or to seawater for 1 day and 7 days, as well as those kept out of water for 1 day. At the end of these periods, poly(A(+))RNA was prepared from the Cl(-)-secreting pectoral skins and also from the outer opercular skins where ion transport is negligible, and analyzed by differential display. We identified four cDNA products expressed differently under various environments as homologues of known genes. A further 34 cDNAs were expressed differentially, but they have no significant homology to identified sequences in GenBank. Northern blots demonstrate that mRNA levels of the actin-binding protein and the platelet-activating factor acetylhydrolase increased in the pectoral skins during seawater acclimation. The mRNA of the 90 kDa heat shock protein was down-regulated in water-deprived and freshwater fish, whose plasma cortisol levels were high. The aldolase mRNA was induced in both skins after desiccation. These four genes may be involved in the environmental adaptations.     

芽孢杆菌α-淀粉酶基因的克隆、表达和酶学性质分析

在仔猪结肠内容物中分离出一株能利用淀粉的芽孢杆菌Bacillussp.WS06,构建了全基因组DNA文库,从中筛选出α_淀粉酶基因amyF,分析测定了其核苷酸序列并进行了表达;其中amyF编码的蛋白有526个氨基酸、分子量为58.6kD;它与已报道的Bacillusmegaterium的α_淀粉酶序列有93%的同源性。经过氨基酸序列比较分析还发现,AmyF含有淀粉酶家族中4个高度保守的酶催化活性区。经多步纯化,重组酶的比活共提高了22.2倍,获得凝胶电泳均一的蛋白样品;经SDS_PAGE检测,AmyF酶分子量为57kD。该酶的最适反应温度为55℃～60℃,酶的最适反应pH为7.0,在温度不超过55℃时,酶活较稳定;AmyF能迅速降解淀粉生成麦芽寡糖,属于内切糖苷酶。     

A broader role for AmyR in <Emphasis Type="Italic">Aspergillus niger</Emphasis>: regulation of the utilisation of <Emphasis Type="SmallCaps">d</Emphasis>-glucose or <Emphasis Type="SmallCaps">d</Emphasis>-galactose containing oligo- and polysaccharides

AmyR is commonly considered a regulator of starch degradation whose activity is induced by the presence of maltose, the disaccharide building block of starch. In this study, we demonstrate that the role of AmyR extends beyond starch degradation. Enzyme activity assays, genes expression analysis and growth profiling on d-glucose- and d-galactose-containing oligo- and polysaccharides showed that AmyR regulates the expression of some of the Aspergillus niger genes encoding α- and β-glucosidases, α- and β- galactosidases, as well as genes encoding α-amlyases and glucoamylases. In addition, we provide evidence that d-glucose or a metabolic product thereof may be the inducer of the AmyR system in A. niger and not maltose, as is commonly assumed.     

IL-2 induces expression of serine protease enzymes and genes in natural killer and nonspecific T killer cells

The expression of serine protease genes was examined in murine NK cells that were purified by panning spleen cells with PMA. Although unstimulated NK cells were cytolytic, they were found not to express the C11 (chymotrypsin-like) mRNA. Culturing these cells in IL-2 (500 to 800 U/ml) for 5 to 7 days induced both the lytic activities and the protease enzymes by 20- to 30-fold. Concomitant to these activation events, the total steady state mRNA of both C11 and HF (trypsin-like) genes were also elevated. The activation of lysis, serine protease enzymes, and C11 and HF mRNA all peaked around day 5 in culture and was dose dependent. In order to exclude the possibility that PMA synergizes with IL-2 in this system, spleen cells from SCID mice, which contained mainly NK cells, were cultured under the same conditions (800 U/ml IL-2, with or without PMA) and PMA did not appear to enhance the expression of these mRNA. Similarly, IL-2 also induced the lytic activities, enzyme levels, and mRNA in the non-Ag-specific T killer cells isolated from spleens of normal mice. Lytic activity of T killer cells was not as high as the NK cells, however, the addition of PHA into the lytic assay resulted in enhanced lysis comparable to that of NK cells. These results showed that lytic activity increased along with protease enzyme levels and mRNA expression in both NK and resting T cells. Therefore, elevated levels of the protease enzymes could be one mechanism involved in optimal lytic activity of IL-2-induced lymphokine activated killer cells.