Regulation of the glucoamylase-encoding gene (glaB), expressed in solid-state culture (koji) of Aspergillus oryzae

JOUFCNAL OF FERMENTATION AND BIOENGINEERING

Vol. 86, No. 3, 301-307.

1998

Regulation of the Glucoamylase-Encoding Gene (glaB), Expressed in Solid-State Culture (Koji) of Aspergilhs oryzae HIROKI ISHIDA,*

YOJI HATA, EIJI ICHIKAWA, AKITSUGU AND SATOSHI IMAYASU

KAWATO,

KOJI SUGINAMI,

Research Institute, Gekkeikan Sake Co. Ltd., 24 Shimotoba-koyanagi-cho Fushimi-ku, Kyoto 612-8385, Japan Received 10 March 1998/Accepted

8 June 1998

Aspergillus oryzae has two glucoamylase-encoding genes, glaA and glaB, the patterns of expression of which are different. Expression of the glaB gene is marked in solid-state culture (koji), but low in submerged culture. To elucidate the induction mechanism of the gZuB promoter in solid-state culture (koji), we employed a fusion gene system using the glaA or glaB promoter and the Escherichia coli uidA gene encoding @glucuronidase (GUS). The expression of glaB-GUS was induced by starch or maltooligosaccharides in a similar manner to that glad-GUS, but other physical factors were found to be required for the maximal expression of the gZuBgane in solid-state culture (koji). The time-course of gZuB-GUS expression in solid-state culture (rice-koji making) suggested that its expression is induced by low water activity (Aw) of the medium and high temperature. When mycelia grown on a membrane under standard conditions were transferred to low-Aw and high-temperature conditions (membrane-transfer culture, MTC), gZuB expression was markedly induced, but that of glad was not. Additionally, gZaB-GUS production was induced in MTC using a membrane with smaller pore size, suggesting that a physical barrier against hyphal extension could regulate glaB expression. Under conditions found to induce giuB expression, namely, starch, low-Aw, high-temperature and physical barriers, approximately 6400 U/mg-protein was obtained, equivalent to that in solid-state culture (koji). In conclusion, glucoamylase production under these induction conditions achieved in MTC reached 274 U/ml-broth, which was equivalent to the level observed in solid-state culture (koji). Northern blot analysis indicated that gZaB expression was induced at the level of transcription 4 h after the transfer to the inducible conditions described above. [Key words: Aspergillus oryzae, glucoamylase,

solid-state culture (koji), reporter

binding

domain

and contains

a larger

due to transcriptional regulation of the glaB gene, by Northern hybridization. It would be of interest to analyse why glaB expression is greater in solid-state culture (koji), and repressed in submerged culture. To identify what regulates glaB gene expression in solid-state culture (koji), measurement of total glucoamylase production is not helpful as it gives no clarification of glaB-specific gene expression. Recent reports have shown that the promoter-GUS fusion and niaD-homologous integration system succeeds in the measurement of the fungal promoter activity (20). Therefore, the promoter fusion system is preferable to determine the individual transcriptional efficiencies of the glaA and glaB genes. We describe the analysis of the glaB gene promoter using the Escherichia coli uidA reporter gene and discuss the important factors affecting glaB gene expression in solid-state culture (koji). To our knowledge, this is the first report on the molecular analysis of Aspergillus in solid-state culture (koji). MATERIALS

shown

to

be

specifically

expressed

in

AND METHODS

Strains, plasmids and culture media A. oryzae (OS1 1013) and its niaD mutant strain (AON-2) were used throughout this study. pNIA2, a 7.7-kb plasmid carrying the 4.5-kb PstI-Hind111 fragment of the niaD gene was selected as the marker in pUC119. E. coli JM109 and pUC plasmids were used for DNA manipulation. The E. coli uidA gene was purchased from

num-

ber of carbohydrate moieties. These results agree with the properties of glucoamylase expressed in solid-state culture (koji) (12, 13). Interestingly, the glaB gene has been

glaB]

culture (koji), but has a very low expression level in submerged culture (12). The inducible glucoamylase production in solid-state culture (koji) was determined to be

Glucoamylase (a-1,6glucan glucohydrolase, EC 3.2. 1.3), which catalyses the release of glucose sequentially from the non-reducing termini of starch or oiigosaccharides, is one of the most useful enzymes in traditional beverage and food industries. In sake brewing, glucoamylase is produced by Aspergillus oryzae during solid-state culture (koji-making) (l), and has the most important role in sake mash as a hydrolytic enzyme (1, 2). A. oryzue glucoamylase is produced in a much higher concentration in solid-state culture (koji) than in submerged culture (3, 4). Many attempts have been made to improve the glucoamylase productivity of A. oryzae in submerged culture by traditional genetic methods (3-9), but there have been no reports describing the high productivity observed in submerged culture equivalent to that in solidstate culture (koji), and the mechanism inducing this higher productivity in solid-state culture (koji) remained unclear. Recently, we demonstrated that A. oryzae has at least two glucoamylase-encoding genes, glaA and glaB (lo12). The glaA gene product in submerged culture has the ability to digest raw starch (10, 11, 13). This glucoamylase, as well as other fungal glucoamylases (14-19), encoded by the glaA, has been shown to possess an Nterminal catalytic domain and a C-terminal binding domain that bind starch. The glucoamylase encoded by glaB has no raw-starch

gene,

solid-state

* Corresponding author. 301

302

.I. FERMENT.BIOENG.,

ISHIDA ET AL.

STRATAGENE (LA Jolla, USA). For submerged culture, the strain was cultured in 30ml of modified Cz-Dox medium, a standard medium (0.3% NaN03, 0.2% KCl, 0.1% KH2P04, 0.05% MgS04.7Hz0, 0.002% FeS04. 7Hz0, and 3% carbon source: glucose or soluble starch) at 30°C for 72 h. Plate culture was performed on CzDox medium containing 1.5% agar at 30°C for 72 h. For solid-state culture (koji), A. oryzae was grown on steamed rice between 32°C and 42°C for 42 h. Rice grains were polished to 70% of their total weight and steamed for 45 min prior to cultivation. For solid-state culture with raw rice, A. oryzae was grown on raw rice saturated with water at 30°C for 72 h. Construction of plasmids for the promoter analysis The plasmids for the promoter analysis were constructed with the promoter-GUS fusion genes. The l.O-kb SaZIX/z01 fragment of the glaB terminator amplified by PCR and the 1.9-kb S&I-XhoI fragment of the E. coli uidA gene were inserted into the SaII site of pNIA2, yielding the plasmid pNGS1 (Fig. 1). When a promoter fragment is inserted into the gap between Pstl and SalI, bglucuronidase (GUS) encoded by the uidA gene is expressed under the control of the inserted promoter. Each promoter region (- 1107 to - 1) derived from the glaA and glaB genes was amplified by PCR using the synthesized oligonucleotide primers. To generate PstI and Safl sites at both ends of amplified promoter fragments, additional sequences, (5’AACTGCAG) and (5’-ACGCGTC GAC) were added to the 5’-end of each upstream and downstream primer, respectively. Each synthesized promoter derived from the gfaA and glaB gene was digested with PstI and S&I, and subcloned into pNGS1 to construct the plasmids pNGA and pNGB, respectively. Transformation and genomic Southern blot analysis niaD mutants of A. oryzae (AON-2) were used as the host strain (13). The transformation of A. oryzae was performed according to the method of Iimura et al. (21). The genomic DNA was prepared according to the method of Tsuchiya et al. (22), and digested by SalI prior to Southern blot analysis. The enhanced chemiluminescence (ECL) detection system (Amersham, UK) was used for signal detection. ucuronidase assay and determination of protein concenZion /%Glucuronidase activity was determined by spectrophotometry using p-nitrophenyl glucuronide, as described by Jefferson et al. (23, 24). One unit was defined as the amount of enzyme that produced one nanomole of p-nitrophenol in a minute at 37°C using a molar extinction coefficient of p-nitrophenol of 14,000 at 430 nm. Protein concentration was measured by the method of Bradford, using bovine serum albumin as the standard (25). Membrane-transfer culture (MTC) To change the water activity (Aw) of the medium in plate culture stepthe membrane-transfer culture wise, we developed (MTC) technique. The transformant was inoculated onto the surface of the nitrocellulose membrane (Advantec Toyo Co. Ltd, Tokyo) on the first plate containing the standard medium. After incubation at 30°C for 2 d, mycelium-grown membrane was transferred onto another plate containing low-Aw medium, followed by additional cultivation at 42°C for 2d. The mycelia on the membrane were collected and subjected to pglucuronidase assay. Enzyme assay of the wild-type strain in MTC The wild-type strain was cultured on a 0.2-pm nitrocellulose

Promoter Insertion Site * sur1JW Y

I

niaD

Sal / Xho

‘Hind111 FIG. 1. Constructed plasmid for promoter region analysis. Methods of plasmid construction are described in Materials and Methods. The arrow represents the promoter insertion sites; PstI and San. The promoter fragment was inserted in the direction of PstI to WI. Sal/Xho site represents the Sal1 site ligated to the XhoI site.

membrane in standard medium and transferred into low-Aw medium containing 50% maltose, for MTC. The standard submerged and MTC media were supplemented with 1% peptone and 0.5% yeast extract. Glucoamylase (GAase) activity was measured according to Iwano et al. (26). Measurement of water activity (Aw) Aw in the medium was determined at 25°C using the apparatus of Rotronic Hygroskop (Rotronic ag. Germany). Northern blot analysis Total RNA was prepared by the method of Cathala et al. (27). About 20 pg of total RNA was resolved by formaldehyde-agarose (1%) gel electrophoresis, transferred to a Hybond-N+ nylon membrane (Amersham, UK) and hybridized with the 1.6-kb EcoRI fragment of the glaB cDNA as a probe. A Gene Image kit (Amersham) was used for the detection. RESULTS Construction of the GUS fusion gene with the gZaA or glaB promoter To elucidate the mechanism of glaB promoter expression in solid-state culture, we constructed two plasmids pNGA and pNGB, containing the glaA and glaB promoters, respectively, fused to the GUS gene. The absence of artificial mutations in the 1.I-kb glaA and giaB promoter fragments synthesized by PCR was confirmed by sequence analysis (data not shown). Introduction of fusion genes into A. oryzae pNGA and pNGB were introduced into the A. oryzae niaD auxotrophs, AON-2, and 6 transformants for each plasmid were obtained. To compare the ,&glucuronidase activities of the transformants, it was most important to select a transformant that harbored the plasmid homologously recombined at the niaD locus of the host genome as a single copy (28). To select such a transformant, Southern blot analysis was conducted (Fig. 2). The niaD gene is located as an 8-kb SalI fragment in the chromosome; therefore, a homologous single-copy integrant will give both 6.5-kb and 13.1-kb signals with a 4.5-kb niaD

REGULATION

VOL. 86, 1998

OF THE gluB GENE

EXPRESSION

IN SOLID-STATE

CULTURE

303

w

123456

1L__ loo0

E

13.lkb6Skb2w

5zg 400 ^M hOO 800 200 0

0 1, Glc

AW FIG. 2. Southern blot analysis of A. oryzae transformants. Ten micrograms of genomic DNA was digested with Sal I and separated by electrophoresis on 0.8% agarose. Following transfer onto a HybondNt nylon membrane, the blot was hybridized with the 45kb &WIHind111 niaD fragment as a probe. Lane 1, AON-2, niaD mutant host strain; lane 2, transformant with pNGS1 containing no promoter region; lanes 3, 4, transformants with pNGA; lanes 5, 6, transformants with pNGB.

fragment is used as a probe in genomic Southern blot analysis. Two clones each for pNGA and pNGB were found to have a single copy of the plasmid integrated through homologous recombination (lanes 3, 4 and 5, 6). BGlucuronidase assays on various culture conditions The P-glucuronidase activities in mycelial extracts of the transformants cultivated in submerged or solid-state culture were measured to analyze the transcriptional efficiency of each promoter (Table 1). In submerged culture with soluble starch as the sole carbon source, GUS production under the control of the glaB promoter was approximately 10% that under the control of glaA. In solid-state culture (koji), however, the GUS production under the control of the glaB promoter was about two hundred-fold greater than that under the influence of glaA. These results supported the previous finding that the glaB gene was specifically expressed in solid-state culture (koji), and not in submerged culture (12). The production of GUS under the control of the glaB or glaA promoter can be considered to represent the promoter expression. The glaA and glaB gene expressions in plate culture were examined (Fig. 3). In plate culture, mycelial growth as aerobic hyphae was similar to that observed in solidstate culture, but the levels of GUS production under the control of glaA and glaB promoters were similar to TABLE

1.

Transformant glaA TF-1 TF-2 gluB TF-I TF-2 B/A

GUS production of two transformants and solid-state cultures Submerged GUS’

1379 118 0.09

GUSa

avr

17 20 4014 3843

19 3929 207

Submerged culture was carried out in 30ml of Cz-Dox medium containing 3% soluble starch. Solid-state culture was performed on steamed rice. The average GUS productions of the two transformants are indicated by “avr”. The glaB/glaA ratio indicated by “B/A” was calculated from average GUS productions of the transformants on submerged and solid-state cultures, respectively. a GUS activity (U/mg-protein).

Glc

Starch

0.970

0.971

0.971

FIG. 3. Comparison of GUS production in submerged and plate cultures. Panel A: submerged culture. Panel B: plate culture. Symbols: q , gluA-GUS production; n , g&?-GUS production.

those in submerged culture. Aerobic hyphae formation was not essential for induction of the glaB promoter, and each culture medium possessed the same water activity (Aw). Effect of carbon sources on glaB gene expression The effects of carbon sources on glaA and amyB gene expressions have previously been determined (29, 30). First, the effect of the carbon source in the culture medium on gene expression in submerged culture was determined (Table 2). The expression of both genes was induced by various maltooligosaccharides, but repressed by glucose. The gene expression of glaB was induced by maltooligosaccharides to the same extent as that of glaA. Second, the sensitivity of the glucose repression was examined (Table 3). Transformants harboring pNGA or pNGB were cultivated in liquid standard medium containing 2% soluble starch and 0 to 10% glucose. Whereas GUS production under the control of both promoters was repressed with an increase in glucose concentration, the glaB/glaA ratios were almost constant irrespective of the glucose concentration. These results suggest that glaA and glaB gene expressions have similar sensitivity to repression by glucose. Finally, the inducibility by raw starch of glaA and glaB gene expressions was investigated (Table 4). In submerged culture, both glaA and glaB gene expressions were induced by raw starch as well as steamed starch. In solid-state culture (koji), raw rice completely abolished GUS production under the control of the glaB promoter in contrast to the results with steamed rice. Although glaB gene expression was induced by starch and repressed by glucose. glaB-GUS production in submerged culture on starch was much lower than TABLE

Solid-state avr

1102 1655 132 103

on submerged

0.969

Starch

Carbon

2.

Effect of various

sources

Glycerol Glucose Maltose Isomaltose Maltotriose Dextrin

Soluble starch Rice powder”

(2%)

carbon

sources

on GUS production

GUS (U/mg-protein) daA

nlaB

39 159 942 844 872 1127 743 800

0.4 5 53 53 69 73 56 84

Each 2% carbon source was used as sole carbon source in submerged culture. a Powder of rice polished to 70%.

304

J. FERMENT.BIOENG.,

ISHIDA ET AL. TABLE 3.

Comparison of sensitivity to glucose repression

Carbon sources Soluble starch (2%) + 2% glucose + 5% glucose + 10% glucose Glucose (2%)

glaA

glaB

839 346 235 124 116

98 37 21 22 7

B/A (%I

12 11 11 17 6

that in solid-state culture (koji) and the induction of gene expression by starch alone does not explain the strikingly high glaB gene expression in solid-state culture (koji). Time-course of the glaB gene expression in solid-state glaB gene expression was suggested to be culture induced not only by starch in the medium, but also by other physical factors in solid-state culture. Figure 4 shows the time-courses of glaA- and glaBGUS production, Aw, temperature and glucoamylase production during solid-state culture (koji-making). The glaA gene was little expressed during the culture up to 50 h. glaB-GUS production was dramatically increased at 30 h, when the Aw of the medium substantially decreased and the temperature of the medium markedly increased. This finding suggests that the Aw and temperature of the medium play a role in the high expression of the glaB gene in solid-state culture (koji). Effects of Aw and temperature of the medium on The effects of physical factors glaB-GUS production such as A w and temperature of the medium on the regulation of glaB gene expression was addressed. For investigation of the effect of Aw of the medium, maltose and sodium chloride were used to decrease the Aw of the plate medium (Fig. 5A). Addition of 0 to 60% (w/v) maltose or 0 to 8% (w/v) sodium chloride to the plate medium gradually reduced the Aw of the medium. Figure 5A shows that glaB-GUS production increased in proportion with decrease in the Aw of the medium. The addition of 50% maltose led to an approximately 5-fold increase of glaB-GUS production in comparison to the addition of 20% maltose. Use of sodium chloride to decrease the Aw of the medium gave similar results to 0.98

I

-300

4

0.97

-250

0.96

-200 $

40 u^ 'L 2

Induction of GUS production by raw or steamed starch Submerged

Strains were cultured in Cz-Dox liquid medium with each carbon sources at 30°C for 2 d.

42

TABLE 4.

GUS (U/mg-protein)

!i -150 ^c 3 -100 g CL!

38

G 36

- 50

glaA glaB

Solid-state (rice)

Raw

Steamed

Raw

Steamed

879 133

822 108

26 9

17 3843

Values represent GUS production (U/mg-protein). For submerged culture, each transformant was incubated in the standard medium containing 2% wheat starch. For solid-state culture, it was cultivated on raw or steamed rice.

those obtained with maltose (data not shown). Figure 5B shows the influence of temperature during plate culture on glaB-GUS production. glaB-GUS production was approximately 3.5-fold enhanced at 42°C compared to that at 30°C and 37°C. Thus, Aw and temperature were indicated to be the positive regulatory factors of glaB gene expression in solid-state culture (koji). However, maximum glaB production conditions, namely, a low Aw and high temperature, resulted in remarkable growth inhibition of the mycelia. Effect of membranes on GUS production When mycelia were grown on a standard plate with a nitrocellulose or nylon membrane, gfaB-GUS production was found to be twofold that on the standard plate media without a membrane, but glaA-GUS production was

q glaJ4 gIaB

0.95

Maltose cont.(%)

20

30

40

growth

++

++

+

(W

50

60

soo-

2 B & a i?

400_

300-

2 3 zoo'5 'S ii loos 0 O-

34

0.92

-0 0

10

20 30 Time(h)

40

Temp(“C)

50

FIG. 4. Time-course of glaB-GUS production in solid-state culture (koji). Symbols: 0, Aw; n , temperature; 0, g/aB-GUS production; V, g/&-GUS production; 0, glucoamylase production.

growth FIG. 5. Effect of Aw and temperature on gfaB-GUS production in plate culture. Aw of plate medium was decreased with the addition of maltose. Panel A: Aw. Panel B: temperature. Symbol: 0, Aw.

VOL. 86, 1998

REGULATION

OF THE glaB GENE EXPRESSION

IN SOLID-STATE CULTURE

305

1000

1I r

200/al-

t_glaB

3

Control

Pore size of membrane (pm)

FIG. 6. Effect of membrane pore size on GUS production. Control represents culture on plates without a membrane. Nitrocellulose membranes of various pore sizes (Advantec Toyo Co. Ltd., Tokyo) were used. Symbols: 0, glaA; m, g/uB.

unchanged (data not shown). Figure 6 shows the effect of pore size of the membrane on GUS production. The production of g&l-GUS was nearly constant both on membranes with various pore sizes and plates without a membrane. On the other hand, gfaB-GUS production increased with a decrease in pore size of the membrane, with a 5-fold higher GUS production on a 0.2-pm membrane than on a standard plate without a membrane. This suggests that glaB gene expression is also enhanced by the physical barrier in addition to other regulatory factors, such as the presence of starch, low A w and high temperature as described above. Effect of membrane-transfer culture (MTC) on the gZaB GUS production It was confirmed that four factors, namely, the presence of starch, low Aw, high temperature and a physical barrier of a membrane with GUS (U/m@ 8KmJ -, 0 6W-

(A)

42’Cshift

tJ,UH,-

A~ 0.935 shift

(B)

q 0.45pm

U.45um 0.2pm

COntrd

GUS (U/u& I(Kil,

Aw 0.935 42’C shift

H

control

0.2pm

42’C shift b’ 0.935 shifl

A~

0.935

FIG. 8. Northern blot analysis of the g/uB gene expression in MTC. A 1.6-kb fragment of gluB cDNA was used as a probe. A. oryzue wild strain, OSI-1013 was cultured in MTC. Lane 1, total RNA from mycelium in constant culture at 30°C for 2 d without membrane; lanes 2-5, total RNA from mycelium after 0, 2, 4, 6 h of additional culture on low-A w plates at 42”C, respectively.

small pore size, induced GUS production under the control of the glaB promoter. To investigate the effects of combinations of the four factors in MTC, mycelia grown on the standard plate media were cultivated under both low Aw and high temperature as described in Materials and Methods. Figure 7 shows the effect of the shift to low A w and high temperature on production of GUS. glaB-GUS production was enhanced by both low Aw (0.935) and high temperature (42”C), and the combination of these factors together with a physical barrier, of a membrane with a pore size of 0.45-pm membrane gave a 30-fold increase in glaB-GUS production over standard culture. When a membrane with a smaller pore size of 0.2~/*m was used in the MTC, glaB-GUS production was further increased with the production of GUS of approximately 6400 U/mg-protein, equivalent to the value obtained in solid-state culture (koji). In contrast, g&t-GUS production was not affected by these factors. These results demonstrated that the four inducing factors were essential for maximal expression of the glaB gene in solid-state culture (koji). Glucoamylase production with MTC As glaB-GUS production in MTC including the four inducible elements reached the level in solid-state culture (koji), glucoamylase production in the MTC reached the level in rice-koji. Then we compared the enzyme production in the MTC system with that in the wild-type strain (Table 5). Comparison of enzyme production under the three culture conditions, the MTC, solid-state (koji) and submerged cultures, revealed that the glucoamylase productivity in submerged culture was lower than that in solid-state (koji), but the MTC system yielded high

42Bc

shift FIG. 7. Effect of inducing factors on GUS production in MTC. Nitrocellulose membranes of two different pore sizes were used (0.45 and 0.2~pm). Panel A: glad-GUS production in MTC. Panel B: glaB-GUS production in MTC. The transformants were inoculated onto nitrocellulose membrane and cultured on a standard plate at 30°C for 2d, followed by transfer onto another standard plate for additional incubation for 2 d. Additional incubation : at 42°C (42°C shift); on a plate containing 50% maltose at 30°C (A ~0.935 shift) ; on a low-4 w plate at 42°C (A w 0.935 and 42°C shift).

TABLE. 5.

Comparison of enzyme productions in MTC and standard cultures GAase

Submerged (U/ml-broth) 2 Solid-state (U/g-koji) 244 MTC (U/ml-broth) 214

AAase

APase

ACPase

375 1250 980

1 144a 211

398 6116 2572

Each culture condition was described in Materials and Methods. GAase, glucoamylase; AAase, tr-amylase; APase, acid protease; ACPase, acid carboxypeptidase.

306

ISHIDA ET AL.

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glucoamylase production (274 U/ml-broth) equivalent to the level in solid-state culture (koji). On the other hand, the productivities of the other enzymes in MTC were higher than those in submerged culture, and in particular, the APase productivity in MTC was increased in comparison with that in submerged culture. Northern blot analysis of gZuB gene expression in the Northern blot analysis was carried out to invesMTC tigate whether the glaB gene expression was regulated at the level of transcription in response to low Aw and high temperature in MTC (Fig. 8). The application and transfer of equal amounts of RNA were verified by ethidium bromide staining (data not shown). No glaB mRNA was detected in the mycelia grown on a standard plate culture (lane l), but it was clearly observed at 4 h after transfer of the medium into low-Aw and high-temperature conditions (lane 4), and was further increased during an additional 2 h cultivation (lane 5). These data demonstrate that glaB gene expression was transiently induced in response to low Aw and high temperature. DISCUSSION Recently, we have characterized a novel glucoamylaseencoding gene (glaB) specifically expressed in solid-state culture (koji) (12). To elucidate the mechanism of glaB gene expression in solid-state culture (koji), we employed the promoter-GUS gene fusion system and measured individual transcriptional efficiency of the glaA and glaB promoters under various culture conditions. This system revealed much information about the regulatory mechanisms of both the glaA and glaB gene expressions and explained why glucoamylase production of A. oryzae is higher in solid-state culture (koji-making) than in submerged culture. GUS production with various carbon sources led us to the finding that glaB expression was induced by maltooligosaccharides and repressed by glucose in the same manner as glaA expression. Interestingly, glaB gene expression was induced by raw starch to the same extent as by steamed starch in submerged culture, although its product had no activity on raw starch (13). Expression of the glaB gene on a standard plate culture was almost the same as that in a standard submerged culture despite the difference in aerobicity of the culture conditions. These results suggest that glaB gene expression in solidstate culture (koji) are directly regulated not only by starch but also by the other factors except for aerobicity. The time-courses of Aw and temperature of the medium on solid-state culture (koji) observed in our study led us to speculate that low Aw and high temperature in the medium play an important role in glaB gene induction in solid-state culture (koji). Another interesting observation was that growth on the membrane with smaller pore size (0.45 or 0.2-pm) resulted in higher glaB-GUS production. This suggests that glaB gene expression responds to the physical barrier against mycelial growth towards the substratum. Recently, Xiao et al. reported that germ tubes of the rice blast fungus, Magnaporthe grisea, recognized the substratum hardness, followed by differentiation into an appressorium (31). Fungi might potentially recognize the hardness of media and there might be several genes that respond to the physical barrier against the mycelium growth towards the substratum. glaB-GUS production in MTC induced by the four inducible factors, namely, the presence of starch, low Aw,

high temperature and physical barriers, increased to the level observed in solid-state culture (koji), suggesting that these factors are necessary and sufficient for glaB gene expression. In koji-making, much attention has been paid to the hardness of steamed rice, depletion of water, and maintenance of high temperature in the later stages of incubation to increase the glucoamylase activity of rice-koji. Interestingly, these conditions, probably established after a large number of trials and errors during the long history of sake brewing, are in good agreement with the factors inducible for gZaB expression. A comparison of enzyme productivities in MTC, submerged and solid-state cultures (koji) was performed. Glucoamylase production in MTC reached 274 (U/mlbroth), equivalent to that in solid-state culture (koji), although it had been difficult problem to improve the productivity in submerged culture up to the level obtained in solid-state culture (koji). From the evidence in this communication, MTC is expected to be a novel solid-state model culture. MTC is thought to be very useful for the analysis of regulatory factors for the production of other enzymes which was much higher in solidstate cultures (koji) than in submerged cultures because culture conditions such as medium composition, Aw, temperature and physical barrier can be readily controlled in MTC. Northern blot analysis showed that glaB gene expression was enhanced at the transcriptional level by transferring cells into low Aw and high temperature conditions in MTC. The glaB gene is expressed in response to the stresses, low Aw, high temperature and a physical barrier, and therefore is a stress-responsive gene. Actually, several consensus heat-shock motifs of Drosophila and Saccharomyces, consisting of arrays of the 5-bp unit AGAAN, are observed in the glaB promoter region (32). Here we have great interest in the biological significance of the stress-response of the glaB gene. It is known that mRNA levels of genes engaged in ATP-generating pathways in cultured cells of rice under different stresses, such as 20% sucrose, salt, cold and nitrogen-starvation stresses, were enhanced (33). In view of this stressresponse, the glaB gene might be urgently expressed for ATP generation on exposure to such stress. There is currently great interest in the stress-responsive mechanism of the gfaB gene at the transcriptional level. Further work is in progress to identify a &-element in response to stress in the gIaB regulatory region. REFERENCES 1. Nunokawa, Y.: Enzymes in sake fermentation. J. Jpn. Sot. Starch Sci., 33, 95-103 (1986). 2. Narahara, H., Koyama, Y., Yoshida, T., Pichangkura, S., Ueda, R., and Taguchi, H.: Growth and enzyme production in a solid-state culture of Aspergillus oryzae. J. Ferment. Technol., 60, 311-319 (1982). 3. Morita, Y., Shimizu, K., Ohga, M., and Korenaga, T.: Studies on amylases of Aspergillus oryzae cultured on rice. I. Isolation and purification of glucoamylase. Agric. Biol. Chem., 30, 114121 (1966). 4. Ohga, M., Shimizu, K., and Morita, Y.: Studies on amylases of Aspergillus oryzae cultured on rice. II. Some properties of glucoamylase. Agric. Biol. Chem., 30, 967-972 (1966). 5. Miah, M. N. N. and Ueda, S.: Multiplicity of glucoamylase of Aspergillus oryzae. Partl. Separation and purification of three forms of glucoamylase. Die Stlrke, 29, 191-196 (1977). 6. Miah, M. N. N. and Ueda, S.: Multiplicity of glucoamylase of Aspergilhs oryzue. II. Enzymatic and physiochemical proper-

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