YEAST

VOL.

7: 337-346 ( I99 1 )

Optimization of Bacillus a-amylase Production by Saccharornyces cerevisiae L. RUOHONEN:. M . PENTTILA* AND S. KERANENt$ Rwomhinunt D.YA Laboratory. b'nirc.rsitJ of Ilelsinki. Valimotie 7 , SF-00380 Helsinki. Finlund

* C'TT. Biotcdmical

Lnhoratorj.. P.O. BO.K202. SF-O2ISl Espoo,Finland

Keccived 7 December 1989: revised 20 November 1990

Production of Bacillus umj~loliyuejucic~n.s a-amylase by Saccharoni~~es cerevisiac. using the multicopy plasmid pAAHS and ways of improving the yields of secreted enzyme were studied. In standard non-buffered medium. a-amylase was rapidly inactivated but stabilization of the pH at 6 led to stable accumulation of a-amylase in the culture medium. Removal of 1100 bp of the upstream sequence of the A D H l promoter present on pAAHS resulted in delayed but increased u-amylase production: 29-fold in selective medium, two-fold in non-selective medium. With the original A DHI promoter. accumulation ofa-amylase in the medium started to level off before the cultures reached stationary phase and was very low when exponentially growing cells were transferred from glucose to ethanol. This coincided with thc appearance of a mRNA larger than the a-amylase messenger. With the shortened promoter, the normal-size (1-amylasem R N A was detected under all growth conditions and a-amylase was efliciently secreted into the medium also late in stationary phase and after transfer to ethanol. Highest total yields of a-amylase were obtained with the short promoter in non-selective glucose-containing medium; this may be explained by the greater final cell density obtained. However. the production of a-amylase per cell mass was higher in ethanol-containing selective medium. Seventy to eighty per cent of the a-amylase activity was secreted into the medium independent of the total amount produced. KEY WORDS

Yeast: ADHl promoter; regulation; hcterologous expression; increased production

INTRODUCTION The ability of the yeast Succharompces cerevisiue to secrete proteins into the culture medium is a potential advantage for production of foreign proteins and has led to studies on expression and secretion of a number of heterologous proteins. Among these are several eukaryotic a-amylases: wheat (Rothstein et ul., 1987), mouse salivary (Thomsen 1983), mouse pancreatic (Astolfi Filho et al., 1986) and human salivary (Nakamura er al., 1986) a-amylase. All these proteins were secreted into theculture medium in a n active form. We have previously shown that Bacillus arnj-lo1iqucfucioti.s u-amylase is efficiently secreted by S. cwcvisiar during the early logarithmic growth phase (Ruohonen et al., 1987). The coding region o f the bacterial gene. including the signal sequence (Takkinen et al., 1983), was placed downstream of the A D H I promoter on the multicopy plasmid +Corresponding author. :Present address: VTT, Riotechnical Laboratory. P.O. Box 202, SF-02 I5 I Espoo. Finland. 0749 SO3X,9I:MOM7 10 S05.00 0 1991 by John Wiley & Sons Ltd

pAAH5 (Ammerer, 1983). However, u-amylase activity rapidly disappears from the culture medium during the late logarithmic growth phase. Here we show that the inactivation of the Bacillus a-amylase activity is due to a decrease in pH of the culture medium and can be prevented by buffering the medium. The A D H l promoter, previously considered to be constitutive, is regulated so that its activity is reduced during growth on non-fermentable carbon sources and in stationary phase (Denis et al.. 1983). It is not clear how this regulation occurs. Activation of a n upstream promoter element creating a longer m R N A from which the A D H I enzyme cannot be translated has been offered as one explanation (Ammerer, 1983). However, Denis Y I ul. (1983) have reported that the longer messenger is functional in ADHI production. Removal of the upstream sequence from the A D H I promoter present on pAAH5 has been shown to abolish the inhibiting effect on A D H I synthesis without affecting the A D H l promoter activity (Beier and Young. 1982). This sequence was deleted from the promoter used

338 for a-amylase expression. Production of secreted a-amylase by both the long and short promoter under different culture conditions is presented. MATERIALS AND METHODS Plasmids, bacteria, yeast strains and media The construction of plasmid YEpaa 1 expressing the a-amylase gene has been described (Ruohonen et al., 1987). Plasmid YEpaa1 (Figure 2) carries the coding region of the a-amylase gene placed between the yeast ADHl promoter and terminator inpAAH5 (Ammerer, 1983). The bacterial strain used for plasmid construction and DNA preparation was a derivative of HBlOl and was grown in L-broth (Miller, 1972)using 100 pg/ml of ampicillin for plasmid selection. The S. cerevisiae strain was DBY746 (a his3A1, leu2-3 leu2-112 ura3-52 trpl-289 cyhR) of D. Botstein. Yeast complete (non-selective) medium YEP-D contained 1% yeast extract (Difco Laboratories, Michigan, U.S.A.), 2% peptone, 2% glucose (Sherman et al., 1983). Yeast minimal medium contained 0.15% yeast nitrogen base (Difco), 0.5% (NH,),SO,, 2% glucose. Synthetic complete medium was yeast minimal medium supplemented with amino acids and nucleotides according to Sherman et al. (1983). SC-Leu was synthetic complete medium lacking leucine. The buffered liquid media contained 3% succinic acid, pH adjusted to 6 with NaOH (Chan and Otte, 1982) and 10 mM-CaC1,. 2% Bacto-agar (Difco) was used to solidify the medium. Yeast was cultivated at 30°C.

L. RUOHONEN, M.PENTTILA AND s. KERANEN

RNAse A (100 pg/ml, 30 min, 37"C), extracted once with buffer-saturated phenol and once with chloroform and then precipitated with ethanol. E. coli was transformed according to Hanahan (1983) and S. cerevisiae according to Ito et al. (1983) or Keszenman-Pereyra and Hieda (1988). Measurement of a-amylase activity

Cells were harvested by centrifugation and the a-amylase present in the supernatant was measured using the Phadebas amylase test (Pharmacia Diagnostics AB, Sweden). To measure cell-bound aamylase the cells were washed once with 0.01 M-Tris-HC1, pH 7.5 containing 0.1 M-NaCl and 0.01 M-CaCl, concentrated 2040-fold into the same buffer and disrupted with a French pressure cell press (Aminco, SLM Instruments Inc., Illinois, U.S.A.). Cell breakage was followed by light microscopy. mRNA extraction and Northern blot analysis

Yeast cells grown in 140 ml of selective medium were harvested by centrifugation and spheroplasted using Zymolyase-100 T (Seikagaku Koguo Co. Ltd, Japan) in 1.2 M-sorbitol. The spheroplasts were pelleted and frozen in liquid nitrogen for storage at - 20°C. The spheroplasts were resuspended into 10 ml of 4 M-guanidiniumthiocyanate (Fluka, Switzerland) and RNA was isolated according to Chirgwin et al. (1979). Three micrograms of total RNA were glyoxylated, electrophoresed in 10 mMsodium phosphate buffer, pH 7.0 in 1% agarose, Determination of cell dry weight and blotted onto a nitrocellulose filter. AlphaTen to twenty-five millilitres of cell suspension amylase-specific mRNA was detected by using as were collected at each time point, the cells were a probe a 1.6 kb internal EcoRI fragment of the washed once with water and resuspended into 2.5 ml a-amylase gene isolated from YRpaa 1 (Ruohonen water. Two millilitres of the cell suspension was et al., 1987) and labelled with [ U ~ ~ P I ~ C using TP pipetted on a watch glass, dried overnight at 110°C the random primer labelling kit of Boehringer and weighed. Mannheim. RNA ladder (0.24-9.5 kb) from Bethesda Research Laboratories, MD, U.S.A. was used as the molecular size markers. Recombinant DNA methods Standard recombinant DNA methods were used (Maniatis et al., 1982). Enzymes were purchased RESULTS AND DISCUSSION from different manufacturers and were used as Efect of culture medium p H on the stability of recommended. DNA fragments were isolated from a-amylase activity agarose gels by using the 'Geneclean' kit (BIO When yeast transformants expressing the a101, CA, U.S.A.). The alkaline-SDS extraction procedure was used for small-scale isolation of amylase gene were grown in either synthetic complasmid DNA from Escherichia coEi (Birnboim and plete medium lacking leucine (SC-Leu) or complex Doly, 1979). During large-scale extraction follow- (YEP-D) medium, a-amylase activity in the culture ing ethanol precipitation the lysate was treated with supernatant was detected only transiently in early

OPTIMIZATION OF BACILLUS a-AMYLASE PRODUCTION BY SACCHAROMYCES CEREVISIAE

B

SC-Leu pH 6 P

I 6

P '

5

-4

I

a

-___ 0

2

4

6

TIME (hl

Figure I . Inactivation of secreted a-amylase in non-buffered yeast culture medium. Yeast cells were grown in non-buffered SC-Leu (A) or in SC-Leu buffered to pH 6 (B). The cell growth was followed by measuring the A,, ( 0 - 0 ) . The pH (A-A) and a-amylase activity (0-0) were measured from the culture medium after removing the cells by centrifugation.

logarithmic phase. This turned out to be caused by the decrease in pH of the culture medium. Buffering of the medium at pH 6 resulted in stable accumulation of secreted a-amylase (Figure 1). In the non-buffered medium, a-amylase activity started to decrease when the pH dropped below 5.6, which in the SC-Leu medium occurred at a cell density of about 1.5 x lo7cells/ml (0.18 mg/ml cell dry weight). In buffered media C a + +ions were added to further stabilize the enzyme (Moseley and Keay, 1970). As shown below (Figures 3 and 5 ) , a-amylase remained stable in the cultures grown in buffered medium in the presence of Ca++ ions for more than 1 week. Only buffered media were used in the following experiments. Several a-amylases have been expressed in yeast but inactivation of the enzyme in the culture medium has not been reported before. Stable accumulation of secreted a-amylases of the mammalian digestive system has been reported (Nakamura et al., 1986; Astolfi Filho et ul., 1986). To keep the human salivary a-amylase active in the yeast culture medium, alkali was added during growth (Matsubara, personal communication). Kunze et al. (1988) studying B. amyloliquefuciens a-amylase and Nonato and Shishido (1988) studying B. stearothermophilus

339

a-amylase were unable to detect enzyme activity in yeast liquid culture medium. In the light of our results this could be due to inactivation of the enzyme by low pH rather than due to lack of secretion or to proteolytic degradation, as suggested (Nonato and Shishido, 1988). In spite of intensive efforts we have been unable to demonstrate any proteolytic activity in yeast culture medium under the conditions in which the a-amylase activity disappeared (Figure 1) even by using the very sensitive hide powder azure assay (Calbiochem@, Behring Diagnostics, CA, U.S.A.) (data not shown). Pretorius et al. (1988) were unable to detect significant amounts of B. amyloliguefaciens a-amylase in synthetic liquid medium, but could detect the activity after transfer of the cells into fresh rich medium. Evidently the pH did not decrease too low under these conditions to irreversibly inactivate the enzyme. Interestingly, in all these reports secretion of the a-amylase from the yeast cells was readily demonstrated by a plate assay, where haloes are formed around yeast colonies producing a-amylase on a starch-containing plate. It seems that the pH on the plate does not decrease below the critical level since our a-amylase-secreting yeast strains are also positive for a halo assay on SC-Leu plates containing 0.1% soluble starch without buffering the medium (data not shown). Deletion of the promoter 5' sequence

The activity of the A D H l promoter is reduced sixto ten-fold at stationary phase and during growth on ethanol as compared to growth on glucose (Denis et al., 1983). While constructing expression vectors containing the A D H l promoter, Ammerer (1983) showed that at stationary phase a new transcription initiation site at about 10OCL1100bp upstream of the normal A D H l initiation site is activated. The longer transcript contains several translation initiation and stop codons preceding the normal A D H l translation initiation site (Bennetzen and Hall, 1982) and thus would not be able to code for ADHI. As deletion of the upstream sequence leaves the A D H l promoter active even in stationary phase and during growth on ethanol (Beier and Young, 1982) we deleted about 1100 bp from the promoter fragment present on pAAH5 (Figure 2). The two a-amylase vectors used in this study have the expression cassette in opposite orientation. In addition, YEpaa4 lacks the pBR322 sequences between BamHI and SphI cleavage sites.

340

L. RUOHONEN, M. PENTTILA

N

YEpaal 13.95kb

S

B

YEpaa4 12.67 kb

Figure 2. Plasmids used for a-amylase expression. T o obtain YEpaal the coding region of the Bacillus a-amylase gene was ligated between the A D H f promoter and terminator in plasmid pAAH5 (Ruohonen et a/.. 1987). The A D H I promoter is present on a 1500 bp fragment of DNA. About 1100 bp of these 5’ flanking sequences were removed to obtain YEpaa4. First the expression cassette of YEpaal was inverted as a BarnHI fragment, followed by removal of a SphI fragment (1 100 bp of A D H I promoter flanking sequences and 187 bp of pBR322 sequences). The restriction enzyme cleavage sites indicated are: B: BurnHI; H: Hindlll; E: EcoRI; S : Sphl.

Production of secreted a-amylase in selective and non-selectivr medium The production of a-amylase using the two vectors described above was studied under selective and non-selective growth conditions by measuring the a-amylase activity secreted into the medium (Figure 3). Higher final yields of a-amylase were obtained with the shortened promoter, as expected. In selective medium the difference was unexpectedly large, the shortened promoter giving 29-fold more aamylase activity. In rich medium the difference was only two-fold. Related to the cell number (cell dry weight), most efficient a-amylase production was obtained with the short promoter in the selective medium. However, both promoters gave higher total activities per volume in rich medium (Table 1). The long promoter yielded 23 times more, and the short promoter two times more a-amylase activity than in selective medium. The higher yields per volume of a-amylase in rich medium may be explained by higher cell densities.

AND

s. KERANEN

Interestingly, there was a clear difference between the two promoters in the appearance of a-amylase activity in the medium and again this difference was more pronounced in selective medium (Fig. 3A). With the long promoter, a-amylase was detectable at a very low cell density and the accumulation started to level off before the cells reached stationary phase. In contrast, with the short promoter, a amylase started to accumulate in the medium only at much higher cell density and continued to be secreted at an exponential rate until late stationary phase. When the orientation of the expression cassette with the long promoter was reversed in comparison with YEpaa I , a-amylase was secreted similarly as when expressed from YEpaa1 (data not shown). Thus, the reduced a-amylase production with the long promoter is not dependent on the orientation of a-amylase transcription on the plasmid. Eficiency of a-amylase secretion We have previously reported that a-amylase is efficiently secreted into the culture medium during early logarithmic phase (Ruohonen et al., 1987). However, the overall level of a-amylase production was very low under those culture conditions. T o determine whether the secretion is still equally efficient when a-amylase production is considerably higher using the short A D H I promoter, the secreted and cell-bound a-amylase activities were measured under low (3 x units/mg dry weight) and high (94 x l o p 2units/mgdry weight) production levels in selective medium. The amount secreted was 78% and 76% respectively, indicating that the secretory capacity of yeast is not limiting the production of a-amylase into the culture medium. Transcription of a-amylase genefrom the long and the short promoter In order to see whether the increased and prolonged production of a-amylase by the shortened promoter could be explained by differences in transcription, the a-amylase-specific transcripts were analysed from exponentially growing and stationary-phase cells (Figure 4). The expected size of the a-amylase mRNA based on the published sequences (Bennetzen and Hall, 1982; Takkinen et al., 1983) is about 2.2 kb. From the short promoter, the expected size of a-amylase mRNA was transcribed at all time points while the long promoter yielded the normal-size messenger only at early

TIME Chl

C”

50

E

6 1

a,

-mm

1.0-

E”

4 b

1

0.1

I

I

I

I

10

50

100

150

0.1

TIME [h) Figure 3. Secretion of a-amylase by yeast expressing a-amylase from the long and the short ADHI promoter. Yeast transformed with either YEpczal (long promoter) or YEpaa4 (short promoter) was grown in selective (A) or non-selective medium (B). Two SO ml cultures in SC-Leu were grown overnight. At A, 0.7 (0.180mg/ml cell dry weight) the cells from one culture were collected by centrifugation and transferred into YEP-D. This time point was taken as 0 h. Thecell dry weight (mg/ml) 0-0 (YEpaa1); A-A (YEpaa4) and secretion of a-amylase activity (units x 10-2/mgcelldry weight) into the medium 0-0 (YEpaal); A-A (YEpaa4) were monitored. The pH was followed throughout and at the end of the experiment was 5.7 in the selective (A) and 6 . 3 in the non-selective medium (B).

L. RUOHONEN, M. PENTTILA A N D s. KERAYEN

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Table 1. Maximal amounts of a-amylase secreted into the culture medium* Promoter

Long

Medium

a-Amylase units x 10- ' Per p e r mg ml cell dry weight

Selective

3

6 I36

Non-selective

Short

14

a-Amylase units x 10 -' Per mg cell dry weight

per ml

160 31 1

86 31

*Final yields in the experiment shown in Fig. 3

logarithmic phase. At late logarithmic phase. when a-amylase production was already levelling off, and later in stationary phase, a longer mRNA was present. These results are in accordance with those

B

A

- 4.4

of Ammerer ( 1983) reported with the construction of pAAH5. Thus, as the cells approach stationary phase a new upstream transcription initiation site is activated and initiation of transcription from the proper A D H I initiation site is abolished. Evidently a-amylase cannot be translated from the longer transcript. At early logarithmic phase more aamylase-specific mRNA was detected with the long promoter than with the short one. This indicates that the different behaviour of the two promoters in production of a-amylase is controlled at the transcriptional level. Production of a-amylase on ethanol using the long and the short promoter

3.2-2.4

2.3-

-1.4

1

2

3

4

1

2

3

4

Figure 4. Transcription of a-amylase-specific scquenccs from the long and the short promoter. Yeast cells transformed with either YEpaal (A) or YEpad4 (€3) were grown in selective medium and harvested at different growth phases. Total RNA was isolated and a-amylase-specific transcripts were detected by Northern blot as described in Materials and Methods. Time points (cell dry weight. mgiml) for RNA isolation were: early lane logarithmic phase (0.10) lane I ; late logarithmic phase (0.64) 2: early stationary phase ( I .o) lane 3; stationary phase ( I .3) lane 4. The numbers on the right indicate the size in kilobases of RNA markers. The numbers on the left indicate the size of the aamylase-specific transcripts in kilobases.

Denis et al. (1983) have shown that the activity of the chromosomal A D H I promoter decreases in stationary phase and when cells are transferred from glucose to non-fermentable carbon source. Using the cloned A D H I gene with the same long and short promoters as we are using but on a single copy plasmid, Beier and Young (1982) have shown that after transfer from glucose- to ethanol-containing medium, ADHI activity decreases when expressed from the long promoter and increases when expressed from the short promoter. I t was therefore interesting to study the production of a-amylase on ethanol. Yeast transformed with YEpaa1 or YEpaa4 was grown in glucose-containing SC-Leu medium until early logarithmic phase and then transferred in the same cell densities into fresh medium containing either glucose or ethanol. Cell growth and aamylase production were followed (Figure 5 ) . In the presence of glucose, a-amylase was secreted into the medium in the expected manner (compare to Figure 3), except that higher levels of a-amylase were obtained with the long promoter (Figure 5A), most

343

OPTIMIZATION OF BACILLUS a-AMYLASE PRODUCTION BY SACCHAROMYCES CEREVISIAE

0 2 0

I

I

100

200

0

20

100

200

TIME Ih) Figure 5. Production of a-amylase on ethanol. Yeast transformed with either YEpaal (long promoter) or YEpaa4 (short promoter) was grown in glucose containing SC-Leu medium. At cell dry weight (mgiml) 0.15 (YEpaal) (A) and 0.18 (YEpaa4) (B), the cultures were divided into two, cells were pelleted by centrifugation and resuspended at the original cell densities in SC-Leu medium containing either 2% glucose or 2% ethanol. The incubation was continued and the cell dry weight (mgiml) 0-0 (glucose containingmedium); A-A (ethanol containing medium) was monitored. a-Amylase activity (units x 10-2/mgcell dry weight) secreted into the medium was measured 0-0 (glucose-containing medium); A- A (ethanol-containing medium).

probably due to the transfer of the cells to fresh medium. On ethanol, cell growth was very much reduced and the final cell density was much lower. As expected, in the case of the short promoter, aamylase production was very efficient, and in fact more a-amylase per cell was produced on ethanol than on glucose (Table 2 ) . The results obtained with the long promoter were somewhat unexpected. As anticipated, after transfer to ethanol-containing medium, a-amylase production was retarded (Beier and Young, 1982). The actual a-amylase activity in the medium between 0 and 20 h was very low, at about the detection limit, and probably due to production in glucose before transfer to ethanol. The relatively high values/mg

dry weight may be misleading due to very low cell densities. After a lag phase of about 30 h, a-amylase activity started to increase in the growth medium and reached almost the same final level as when expressed from the short promoter. The a-amylase-specific mRNAs were analysed from a similar experiment (Figure 6). With the short promoter the normal-size a-amylase messenger was detected both on glucose and after transfer to ethanol, as expected. The weak band seen at about the same position as the longer messenger after transfer to ethanol must initiate outside the expression cassette since it is about 600 nucleotides longer than the entire expression cassette, including the promoter and terminator sequences (Bennetzen and Hall,

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L. RUOHONEN, M. PENTTILA AND S. KERANEN

Table 2. Maximal amounts of a-amylase secreted into the culture medium in glucose and after transfer to ethanol.* Promoter

Long

Short

a-Amylase units x per mg cell dry weight

a-Amylase units x Per Per mg ml cell dry weight

Carbon soGrce

Per ml

Glucose Ethanol

154

15

7 253

56 357

147

253

*Final yields in the experiment shown in Fig. 5.

1982; Ammerer, 1983; Takkinen et al., 1983). With the long promoter on glucose, the normal-size messenger was dominant but the longer transcript was also seen. On ethanol, 21 h after transfer from glucose, a-amylase secretion into the medium had already re-started after the initial lag period in this experiment (data not shown). Thus the presence of the normal-size messenger could be anticipated. However, the longer transcript was seen as a much stronger band (Figure 6). Later on, when a-amylase production became more efficient, the normal messenger became more abundant and the amount of the longer transcript was greatly reduced. Thus, transfer of exponentially growing cells to ethanol allows transcription initiation at the upstream initiation site. One could speculate that the upstream initiation site or promoter is repressed by glucose and therefore derepressed on ethanol, or at stationary phase when glucose has been exhausted. The reappearance of a-amylase production with concomitant disappearance of the longer transcript after prolonged growth on ethanol was somewhat surprising. Whether this can be explained by efficient enough gluconeogenesis under these conditions remains to be shown. However, the overall situation must be more complex than glucose repression of the longer transcript only, since the normal A D H l transcription initiation site is active much earlier on the long than on the short promoter (Figures 3A, 4), suggesting that positive regulatory sequences of ADHI are present in the deleted part of the original promoter. Recently, Tornow and Santangelo (1990) have shown that efficient transcription of A D H l requires the UAS,, consensus sequence located at -628 to -616 with respect to the + 1 mRNA start site. This cis-acting element is recognized by the DNA-binding protein T U F (Huet

1987), which has been proposed to be needed for efficient transcriptional activation of the glycolytic genes. A 25-fold reduction in A D H l transcription was observed if UASRPGwas deleted (Tornow and

et al., 1985; Leer et al., 1985; Huet and Sentenac,

Santangelo, 1990).

3.2-

2.3-

1

2

3

1

2

3

Figure 6. Transcription of a-amylase-specific sequences after transfer to ethanol. Yeast transformed with either YEpaal (A) or YEpaa4 (B) was grown and transferred to ethanol-containing medium as described for Figure 5 . Total RNA was isolated and a-amylase-specific transcripts were detected by Northern blotting as described in Materials and Methods. Lane 1: RNA isolation from glucose-grown cells (cell dry weight 0.18 mg/ml); lane 2: RNA isolated 21 h after transfer to ethanol (cell dry weight 0.21 mg/ml); lane 3: RNA isolated 89 h after transfer to ethanol (cell dry weight 0.38 mgiml). a-Amylase activity (units x lo-’)/ mg cell dry weight: (A) lane 1: 7.8; lane 2: 1.7; lane 3: 25; (B) lane 1: 2.6; lane 2: 8.9; lane 3: 102.

OPTIMIZATION OF BACILLUS a-AMYLASE PRODUCTION BY SACCHAROMYCES CEREVISIAE

This cis-acting element is deleted in our shortened ADHI promoter construction. It would be interesting to see whether addition of this element to the short promoter would further increase production of a-amylase. However, as the UAS,,, has no effect on the carbon source regulation of A D H l promoter (Tornow and Santangelo, 1990)we must have removed an additional regulatory sequence having a positive effect on glucose-grown cells at low cell densities. Santangelo and Tornow (1990) also suggest glucose-inducible regulation of A D H l transcription by a hypothetical trans-acting regulatory protein, which would bind to a sequence downstream from -403 on the ADHI promoter (with respect to the 1 mRNA start site). The deletion in our short promoter extends to - 377 and may affect this glucose-mediated induction and consequently a-amylase production. The question remains why a significant amount of a-amylase is produced from the long promoter upon prolonged incubation on ethanol.

+

CONCLUSIONS Bacillus a-amylase was used to study production of a secreted heterologous protein by S. cerevisiae. Several parameters affected the final yields of active enzyme, most crucial of them being the pH of yeast culture medium. Removal of inhibitory sequences preceding the ADHI promoter (Berier and Young, 1982; Denis et al., 1983; Ammerer, 1983) allowed a-amylase production until late stationary phase and to much higher yields than using the long promoter construction both in selective and non-selective medium. Highest final yields per culture volume were obtained in rich medium and these were 50fold higher than those obtained with the long promoter in selective medium. However, when the production of a-amylase was calculated per cell mass, more efficient production was obtained in selective medium. Also the carbon source affected a-amylase production: with both promoters ethanol gave higher final yields per cell than glucose in prolonged cultivations. The easily scorable a-amylase was used as a marker to study transcription from the A D H l pror moter on pAAH5. Reduction in ADHI synthesis and appearance of a longer messenger under defined growth conditions have been reported before (Beier and Young, 1982; Ammerer, 1983; Denis et al., 1981, 1983) but it has not been rigorously shown that these occur simultaneously, as shown in the

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present study. The contradictory results reported, on whether ADHI can be translated from the longer messenger (Denis et al., 1983; Ammerer, 1983) could be due to strain differences. Our results show that functional a-amylase cannot be translated from the longer messenger transcribed from the plasmidborne A D H l promoter. Removal of the upstream sequence of the A D H l promoter on pAAH5 delays the initiation of a-amylase transcription, but once commenced allows it to continue until late stationary phase. It would be interesting to know whether the upstream sequences have evolved to control the A D H l expression under different growth conditions and are present in all yeast strains and not only in the strain from which the A D H l promoter has been isolated. Previous studies have indicated that the ADHI transcription is regulated in carbon sourcedependent manner. However, a detailed analysis of the transcription during different states of growth has not been reported before. Our present results suggest that the mode of transcription is dependent not only on carbon source but also on additional regulatory aspects which vary during growth. ACKNOWLEDGEMENTS We thank Ilkka Palva and Alko Ltd for the aamylase gene and Benjamin Hall for the yeast expression vector pAAH5 and for fruitful discussions concerning its use. Ms Riitta Lampinen is acknowledged for excellent technical assistance. This work was financially supported by The Academy of Finland and Nordic Yeast Research Program. L.R. was a recipient of the Neste Foundation Fellowship. REFERENCES Ammerer, G. (1983). Expression of genes in yeast using the ADCI promoter. Methods Enzyrnol. 101, 192-201. Astolfi Filho, S., Galembeck, E. V., Faria, J. B. and Schenberg Frascino, A. C . (1986). Stable yeast transformants that secrete functional a-amylase encoded by cloned mouse pancreatic cDNA. BiolTechnology 4, 3 11-3 15. Beier, D. R. and Young, E. T. (1982). Characterization of a regulatory region upstream of the ADR2 locus of S. cerevisiae. Nature 300,724-728. Bennetzen, J. L. and Hall, B. D. (1982). The primary structure of the Saccharornyces cerevisiae gene for alcohol dehydrogenase I. J . Biol. Chern.257,3018-3025. Birnboim, H. C . and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7 , 1513-1 523.

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