Introduction
The useful protein (enzyme) produced by gene manipulation technology is used in fields broad now, such as a medicine use and an industrial  way. Thus, recombinant protein has brought modern society many benefits. Then, various protein expression systems by the microorganism, the insect and the plant host are developed in order to produce such useful protein more efficiently. The high expression system by filamentous fungi which authors developed is also one in it. There are mainly two merits which use In the 1st, Aspergillus species as a host. Aspergillus species which essentially produces various the protein enzymes that is productivity is superior to other hosts. As the 2nd, it is estimated that A. oryzae and A. niger which are used for manufacture of Japanese fermentation food, such as sake, miso, and soy sauce, are safe also as a host of protein production 1 ). Recently, the report of production various recombinant proteins by the of Aspergillus species host is increasing notably2,3). And industrial use of some enzyme has already been carried out. When producing the target protein by genetic engineering, the production efficiency is regulated by various factors, such as promoter activity, translation efficiency, glycosylation to the expressed protein, the copy number of a gene, degradation by fungal target and However, the most important proteases. factor it how increase the in is to transcriptional level. From this viewpoint, discovery of a strong promoter is important. Recently, also in Aspergillus oryzae, there is movement toward extensive acquisition of the promoter using EST (expressed sequence tag) analysis data and acquisition of the intellectual property rights about the promoter.   However, since it depends for production of useful enzyme on the selected promoter activity and the expression characteristic in this case, the promoter who uses it has chosen according to the purpose of target enzyme.    In this    review, it introduces about the high expression system in Aspergillus species developed by using the improved promoter containing cis-element of the amylase genes 4 , 5 ).
Regulatory mechanism for the A. oryzae
amylase genes
A. oryzae produces industrially valuable Taka-amylase (TAA)6), amylases, such as glucoamylase (GLA)7) and α-glucosidase The expression of amyB, glaA and (AGL)8) agdA genes encoding TAA, GLA and AGL respectively is induced by starch or maltose and repressed by glucose as a carbon source at the transcriptional level. A comparison of the promoter regions of the Aspergillus amylase genes has indicated that are four highly conserved sequences, designated Region I, II, IIIa and IIIb (Fig. 1) 9). The function of these consensus sequences has been investigated by deletion analysis in the agdA promoter (PagdA).  This result suggests that Region IIIa is a functional element essential for high-level expression and maltose induction. Deletions of Region IIIb have suggested that this element contains sequences that might be involved in enhancing expression in conjunction with region IIIa.  Accordingly, multiple copies of the fragment comprising with Region IIIa, b were introduced into PagdA to enhance promoter activity and to determine the common regulatory mechanism for the expression of the A. oryzae amylase genes 10 ). The improved promoter (PagdA142) carrying 12 tandem repeats of Region IIIa,b resulted in a significant increase in promoter activity. Moreover, the transformant carrying the agdA gene under the control of the improved agdA promoter resulted in efficient overproduction more than 100-fold AGL activities of the recipient strain. Interestingly, expression of the amyB and glaA genes in the transformant was strongly repressed. Since this phenomena was observed at the transcriptional level, these results suggest that the fragment comprising Region consist of one or more cis acting IIIa,b sequence(s).    Recently, a gene, amyR which
encodes a transcriptional activator of the amylase genes has been cloned from A. oryzae11,12), A. niger and A. nidulans13,14).  And the CCAAT binding protein which has an influence on the overall expression of several fungal genes has been also detected and cloned from A. nidulans15~17) and A. oryzae18). Now, as shown in Fig. 2, regulatory mechanism model for the A. oryzae amylase genes is demonstrated. The amyR protein, AMYR, which involved in maltose induction and high-level expression, binds to Region IIIa. The A. oryzae CCAAT binding protein, AoCP, consists of the HAP complex (HAPB, HAPC and HAPE) 18) , and binds to Region IIIb. In the wild-type strain, the expression of the A. oryzae amylase genes is enhanced by two regulatory mechanisms of Region IIIa-AMYR and Region IIIb-AoCP. However, in the AGL-overproducing transformant, the transcriptional activator such as AMYR and AoCP in interacting with Region IIIa,b are common to these amylase genes and that the of regulatory proteins titration by the introduction of multiple copies of Region IIIa,b leads to a reduction in TAA and GLA production.
Development of high expression system
with the improved promoter
We demonstrated that Region III comprising two cis elements is important on the regulatory mechanism for A. oryzae amylase genes. Furthermore, improvement of promoter activity has been exhibited by the introduction into the agdA promoter of multiple copies of the Region III. In this chapter, we describe about the effects of Region III insertion and utilities of the improved promoter in various Aspergillus species.
(1) The effects of Region III insertion
High-level expression signals such as the amyB and glaA promoters are frequently used for the over-expression of homologous and heterologous genes in a variety of filamentous fungi. If the effects of Region III insertion are observed other promoters, in the the application of Region III could extend to the efficient production of enzymes in filamentous fungi. Accordingly, multiple copies of the fragment comprising Region III were introduced into the glaA promoter (PglaA) and No. 8 promoter (P-No8) 19 that was high-level
expression signal isolated from A. niger. The promoter activities of the improved glaA promoter (PglaA142) and the improved No. 8 promoter (P-No8142) increased to be 4-6 folds higher than that of the original promoter (Fig. 3). And these improved promoters resulted in 3-fold higher activities than the improved agdA promoter (PagdA142)4, 5) Moreover, introduction of the multiple copies of Region IIIa,b into the enoA promoter (PenoA) 201 that was one of the most strongly expressed genes in A. oryzae glycolytic genes
The fragment of 12 tandem repeats of a) Region III was introduced into the styl site of enoA promoter. b) GUS was activity of transformant measured as described in Fig. 3. also resulted in the improved promoter (PenoA142) more than 20-fold activities of the native promoter (Table 1)²¹). And the activity of PenoA142 increased to be 2.5-fold higher than that of P-No8142. This improved promoter is probably most strong in A. oryzae. In addition, introduction of the reverse fragment into P-No8 and PenoA also resulted in a significant increase. These results suggest that Region III not only is useful to improvement of the promoter of amylase genes, but also that of various from Aspergillus species. promoters The insertion of a cis-acting sequence into the promoter region is an effective means of increasing promoter activity. (2) Utilities of the improved promoter in various Aspergillus species To confirm utilities of the improved promoter in various Aspergillus species other than A. oryzae, the activity of improved No. 8 promoter (P-No8142) was compared with the intrinsic No. 8 promoter (P-No8) in A. niger and A. usamii. The P-No8142 activity increased to be 2- to 3-fold higher than that of the P-No8 both hosts. This result suggests that the improved promoter using Region III is useful to
the over-expression of in various genes Aspergillus species as a host. Moreover, the increasing activity of the improved promoter shows the presence of a common regulatory mechanism interacted with Region III in Aspergillus species. In fact, the amyR gene has been cloned from A. niger and A. nidulans.
Molecular breeding of useful enzyme
overproducing transformant in
Aspergillus species
It has constructed the expression vector that consist of the improved promoter, the agdA terminator, multi cloning site and the niaD encoding nitrate reductase gene22) as a selectable marker (Fig. 4). We have obtained several overproducing transformants of useful enzyme by the transformation system using Aspergillus species niaD mutant as a host. In chapter, we describe about this the overproducing transformant of nuclease S1 (NUC) and 1, 2-α-D-mannosidase (MSD). A. oryzae NUC is one of the nuclear degradation enzymes and useful enzyme as a reagent of gene manipulation. However, since the expression level of nucS gene²³) encoding NUC is very low in A. oryzae wild-type strain, using the improved agdA promoter (PagdA142), NUC-overproducing transformants the are obtained in A. oryzae. In these transformants, the NUC activities increased to be 1000-fold higher than that of A. oryzae host strain, and concomitant reduction of the TAA and GLA activities were remarkably observed. The result of SDS-PAGE analysis showed that the amount of NUC is more than 80% yield of the total secreted proteins in the submerged culture. Similar phenomena have been also observed in wheat bran solid-state culture. In addition, NUC production was more efficient in wheat bran solid-state culture (up to 20-fold) than in the submerged culture, resulting that the productivity was more than 1.0g/kg-wheat bran. In the next place, to compare the ability of production several host, NUC the in P-No8142::nucS fusion gene was introduced into A. oryzae, A. usamii, A. niger and A. saitoi. Each transformant carrying similar multiple copies showed high-level and same degree both solid-state and the production the culture. This results submerged that the examined four Aspergillus species is excellent as a host. SDS-PAGE profile of A. niger transformant in the submerged culture showed in Fig. 5A. In the second, we describe about overproducing transformant of A. saitoi 1, 2-α-D-mannosidase (MSD) which is intracellular enzyme and useful as a reagent of glycotechnology. The msdS gene encoding MSD was cloned by Ichishima et al 24 ). The fusion gene which was constructed with the signal sequence of aspergillopepsin I gene, apnS, and the msdS fragment, was introduced into A. oryzae using the improved No. 8 promoter (P-No8142)25). The secreted MSD of transformant remarkably high level was We also (0.5-0.6g/l). obtained the MSD-overproducing transformants in A. saitoi host. Those transformant showed high-purity and overproduction to more than 2000-fold in comparison with A. saitoi wild-type strain (Fig. 5B). Recently, Kawaguchi et al. have reported transformants of A. that the aculeatus ß-mannosidase achieve a remarkable high (more than  2.5g/l) production using the P-No814226). These results demonstrate that the high expression system using the improved promoter is useful to efficiency overproduction of low level enzymes. Moreover, the reduction in the production of amylase, which is a major secretory protein in  Aspergillus species, resulted in an increase in the specific activity of the target enzyme. Therefore, the purification of target enzyme is easy and price down of production costs is possible.
Future development
Whole genome sequence various of organism such as plant, mammalian and microorganism have been determined in the past several years so that it may be represented by human genome analysis. In A. oryzae, it was vigorously promoted in our country, and first, EST analysis was carried out in about two years after 1999, and the partial sequences of about 5000 genes was determined. Then, draft sequence completion of whole genomes was released in January, 2002, and it was shown in A. oryzae that about 13000 genes exist² ⁷ . A. oryzae strains and their products have been used in food and the food processing industry and have resulted in a so-called GRAS (generally regarded as safe) status 1 ). Therefore, A. oryzae is safe also as a gene resource and the usefulness is high. We are continuing research of Aspergillus species from ten years or more before, and have succeeded in development of an effective high expression have system. Moreover, we also the participated in project of above-mentioned EST analysis and genome analysis, and can receive also about the newest gene information. That is, we have the core technology about recombinant protein production and gene information on A. oryzae, and consider positive business deployment using technology and information. We want to strengthen joint development between companies, and cooperation with industrial, administrative and academic sectors, and to get a business chance for that purpose, more than former. Please tell us, if some interested in our technology and business plan.
Conclusion
Now, gene manipulation technology is used not only in the overproduction of useful protein but in various fields. For example, these technologies are also used in the biomass, environmental preservation and the ecology field, and these fields will develop greatly from Only one being regrettable is that a now on. hurdle is still expensive about the application to food. This cause is not being obtained in sufficient public acceptance about safety. In order to improve this situation, industrial, sectors administrative and academic pull together, and much more honest efforts are We want believe that required. to an understanding application gene of of manipulation to food technology or fermentation industry is obtained in the near future. In this study, we have shown the presence of oryzae regulatory common a in A. mechanism interacted with Region III in the expression of the amylase genes, amyB, glaA, and agdA. Moreover, we have demonstrated the advantage of over expression of genes controlled by the improved promoter using Region III in various Aspergillus species. However, this high expression system by Aspergillus species probably further is improvable. For that purpose, it is necessary to analyze a series of regulatory mechanisms, such as transcription, translation, glycosylation, secretion and proteolytic degradation, in detail. We will try the development of a more efficient high expression system based on new finding in the near future. And we want to contribute to development of biotechnological industry using this expression system.