Current Genetics

Current Genetics (1984) 8:471-475

© Springer-Verlag 1984

Short Communication

Expression of the cloned endo-l,3-1,4-fl-glucanase gene of Bacillus subtilis in Saccharomyces cerevisiae Edward Hinchliffe and Wendy G. Box Research Department; Bass PLC; 137 High Street; Burton on Trent DE14 1JZ; Great Britain

Summary. A cloned endo-l,3-1,4-fl-glucanase gene from the Gram-positive bacterium B. subtilis has been located by deletion analysis on a 1.4 kb PvuI-ClaI DNA fragment. This gene has been sub-cloned in the yeast LEU2 vector pJDB207 to produce a hybrid plasmid designated pEHB9. pEHB9 has been transformed to S. cerevisiae and shown to direct the synthesis of an endo-l,3-1,4-fl-glucanase in yeast. The fl-glucanase activity was low and could only be detected in crude cell extracts of yeast harbouring pEHB9. Key words: fl-Glucanase - Expression - Heterologous DNA - Yeast

tional enzyme in E. coli (Hinchliffe 1984). The cloned fl-glucanase gene was shown to encode an enzyme specific for the hydrolysis of barley fl-glucan, and was found to be predominantly extracytoplasmic in location in strains of E. coli (Hinchliffe 1984). Here we describe the molecular localization of the/3-glucanase gene within the 3.5 kb E c o R I fragment, the sub-cloning of the gene to a yeast/E, coli shuttle vector and its subsequent introduction into S. cerevisiae. We show that the fl-glucanase gene is capable of encoding a biologically active protein in S. cerevisiae and that the enzymic activity is characteristic of that found in culture supernatants of B. subtilis and E. coli.

Materials and methods

Introduction High levels of fl-glucan in brewers wort can lower the rate of beer filtration (Eyben and Hupe 1981) and cause the formation of undesirable hazes and gelatinous precipitates in finished beer (Gjertsen 1966; Letters 1969, 1977). Although yeasts produce several different types of fl-glucanase, none are able to hydrolyse fl-1,3-1,4 linked glucan found in barley (Abd-E1-Al and Phaff 1968). Problems associated with fl-glucan in beer can be alleviated by the application of commercial microbial enzyme preparations at the beginning of fermentation or during beer storage (Enkenlund 1972; Leedham et al. 1975). Nevertheless, it would be advantageous if fl-glucan could be degraded by brewers yeast during fermentation. In pursuit of this objective, we have recently cloned a 3.5 kb E c o R I DNA fragment carrying an endo-l,3-1,4-fl-D-glucanase gene from B. subtilis, that is expressed as a funcOffprint requests to: E. Hinchliffe

Organisms and plasmids. The bacterial strains used wereB, subtilis strain NCIB 8565 (National Collection of Industrial Bacteria) and E. coli K-12, strain HB101 (hsdS, leu, pro, lac, gal, strA, thi, recA) (Boyer and Roulland-Dussoix 1969). The strain of S. cerevisiae was X4003-5B (a, ade-1, his-4, leu-2, met-2, trp-5, ura-3, gall) (Yeast Genetic Stock Centre, University of California, Berkeley). The construction of plasmids pEHB3 and pEHB5 carrying the cloned fl-glucanase gene have been described previously (Hinchliffe 1984). The yeast vector used was pJDB207; this plasmid contains the yeast LEU-2 gene and can be introduced by transformation into both E. coli and yeast, replicating in both organisms as an autonomous extrachromosomal element (Beggs 1981). Media and growth conditions. The media used for the growth of bacterial ceils (described by Hinchlfffe; 1984) was supplemented where appropriate with the antibiotics ampicfllin and tetracycline at final concentrations of 25 pgml- 1 and 15/~gml- 1, respectively. The media used for the growth of yeast cells were YED (1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose, solidified with 2% w/v agar) and YNB, a buffered minimal medium described by Johnston et al. (1977).

472

E. Hinchliffe and W. G. Box: Expression of a prokaryotic gene in yeast

Plasmid p~B3

A BE I II q Tc

E BgAP / ~ l

CE ~l

pEFIB301

P I ~p

Ap Tc BG R R +

R

S

+

R

S

+

S

R

-

R

R

+

R

S

-

R

S

-

pEHB508

S

R

+

pEHB50/,

R

S

+

pEHB304

--

pEHB306 EC It"

pEHB5

Enzyme preparations from S. cerevisiae. For the preparation of crude cell extracts from yeast, ceils were grown in YNB buffered minimal medium supplemented with the appropriate amino acids, harvested in late logarithmic phase and disrupted using a Braun homogenizer. Glass beads and cell debris were removed by 8,000 x g for 10 min, and the supernatant was recentrifuged at 10,000 x g for 30 min and dialysed against 2 x 21 of 0.1 M phosphate/citrate buffer pH 5.6 at 4 °C.

P~I~ E I

I

'To pEHB506

Transformation of E. coli and yeast. Yeast cells were transformed as described by Beggs (1978). The method of Wilson and Murray (1979) was used to transform E. coli cells.

Results Molecular location o f the ~-glucanase gene

pEHB501

--

Fig. 1. Molecular location of the ~-glucanase gene within the cloned 3.5 kb EeoRI fragment of B. subtilis NCIB 8565 DNA. Restriction sites for the enzymes: AvaI (A), BamHI (B), BgllI (Bg), ClaI (C), EcoRI (E) and PvuI (P) are indicated in the vector ( - - ) and B. subtilis ( m ) DNA sequences. The genes specifying resistance to the antibiotics ampicillin (Ap) and tetracycline (Te) contained within the vector DNA are indicated by arrows. The phenotypic notation R and S refers to resistant and sensitive respectively; the 13-glucanase(BG) phenotype was determined as being either positive (+) or negative (-) on the basis of the plate screening procedure for /3-glucanase activity associated with single colonies

Assay and plate screening for #-gIucanase activity. These were as described by Hinchlfffe (1984), with the exception that reducing sugar assays were performed at 40 °C at various pH's. Citratephosphate buffers were used from pH 4.0 to 6.8 and phosphate buffers from pH 6.8 to 7.6. Test for #-lactamase activity. The ~-lactamase activity of yeast transformants were assayed by the method of Chevallier and Aigle (1979). Protein estimates. These were made from the absorption of UV light at 230 and 260 nm according to Kalb and Bernlohr (1977). Isolation ofplasmid DNA. This was isolated by caesium chloride/ ethidium bromide equilibrium density centrifugation of cleared cell lysates using the method of CleweU and Helinski (1969), with the modifications of Zahn et al, (1977). Restriction, ligation and electrophoresis of DNA. DNA restriction and ligation was performed as recommended by the suppliers (Bethesda Research Laboratories, Cambridge, UK and New England Biolabs, Bishops Stortford, UK). Analysis of restriction digests was by agarose gel electrophoresis (Helling et al. 1974).

The recombinant plasmids pEHB3 and pEHB5 carrying the 3.5 kb E c o R I DNA insert have two restriction sites for each of the enzymes AvaI, ClaI, PvuI and BgllI/ BamHI, one of which is contained within the insert DNA and the other in the vector DNA (Hinchliffe 1984). This observation was used to map more precisely the/3-glucanase gene in the 3.5 kb E c o R I fragment. By making deletions from within the E c o R I fragment extending into either of the adjacent ampiciUin- or tetracycline-resistance genes, it was possible to isolate deletion derivatives as being either ampicillin or tetracycline sensitive, respectively. These derivatives were then screened for /3-glucanase activity (Fig. 1). It can be seen that the /3-glucanase gene is located on a 1.45 kb PvuI-EcoRI DNA fragment carried by plasmid pEHB508 (Fig. 1). Since the ClaI deletion derivative pEHB504 retains ~-glucanase activity it can be assumed that the active ~-glucanase gene resides within the 1.4 kb PvuI-ClaI DNA fragment.

Construction o f a yeast strain containing the ~glucanase gene o f B. subtilis To introduce the ~-glucanase gene of B. subtilis into S. cerevisiae, we made use of the shuttle vector pJDB207, that can replicate in both E. coli and S. cerevisiae. As BgllI does not interrupt the integrity of the/3-glucanase gene (Fig. 1), it was decided to sub-clone the 3.5 kb E c o R I fragment, by in vitro rearrangement, into the BamHI site of pJDB207 (Fig. 2). The resultant plasmid, designated pEHB9, was phenotypically ampicillin resistant, tetracycline sensitive and t3-glucanase positive in E. coli. pEHB9 was then introduced into the leu-2 recipient of S. cerevisiae, X4003-5B, selecting for L E U + transformants on minimal medium. One L E U + transformant was isolated which when assayed for ~-lactamase activity proved positive. Confirmation that the strain was a plasmid transformant was obtained by observing

473

E. Hinchliffe and W. G. Box: Expression of a prokaryotic gene in yeast B

the segregation of the L E U + character by growth on non-selective medium. The L E U + plasmid transformant was designated X4003-5B (pEHB9) and a leu- segregant, X4003-5B (pEHB9-). Plasmid pJDB207 was similarly transformed to X4003-5B to act as a control strain in subsequent enzyme assays. E B Expression o f endo-l ,3-1, 4-fl-glucanase activity in S. cerevisiae

CE EcoRI,

Ligase

E

E ~/Bg

E

S. cerevisiae does not contain endo-l,3-1,4-fl-glucanase activity. However, Table 1 shows that X4003-5B acquires endo-l,3-1,4-fl-glucanase activity after transformation by pEHB9. The control strain X4003-5B (pJDB207) and the leu- segregant X4003-5B ( p E H B 9 - ) do not contain endo-l,3-1,4-fl-glucanase activity. Therefore the presence of endo-l,3-1,4-fl-glucanase activity in yeast appears to be correlated with presence of the hybrid plasmid, pEHB9, carrying the B. subtilis fl-glucanase gene. The low level of fl-glucanase detected is apparently confined to the cell associated enzyme preparations, no activity being detected in freeze dried culture supernatants o f X4003-5B. (pEHB9). fl-Glucanase activity could not be detected in single colonies of X4003-5B (pEHB9) using the fl-glucanase plate screening procedure.

P

C P

A

Fig. 2. Construction of the hybrid plasmid pEHB9, carrying the cloned "/~-glucanasegene. ( m ) B. subtilis, (V---q) yeast and ( ) vector DNA sequences, ( c ,) position of the fl-glucanase gene. The relative location of restriction sites are indicated for the enzymes: AvaI (A), BamHI (B), BgllI, ClaI (C), Pvul (P) and BamHI-BgllI hybrid site (B/Bg). Ligase treatment following EcoRI digestion of pEHB3 was performed under dilute DNA concentrations, thus favouring circularization of the two products of EcoRI digestion. The restriction enzymes BamHI and BgllI generate mutually compatible cohesive ends which can be successfully ligated to form a hybrid site (B/Bg) which is not recognised by either of the parental enzymes. BarnHI and BgllI digestion and ligation were performed at higher DNA concentrations, favouring recombination of the rearranged B. subtilis DNA in the tetracycline gene of pJDB207. Transformants were selected in HB101 as being ampicillin resistant, tetracycline sensitive and fl-glucanase positive

Comparison o f the endo-l,3-1,4-fl-glueanase expressed in yeast with the B. subtilis and E. coli fl-glucanase Since the enzyme activity associated with crude cell extracts of yeast is correlated with the presence of pEHB9, it is not unreasonable to assume that activity is attributable to expression of the B. subtilis fl-glucanase gene in yeast. We compared the activity of the fl-glucanase enzyme isolated from culture supernatants of B. subtilis and E. coli, with that isolated from X4003-5B (pEHB9). The fl-glucanase activity is considerably reduced in yeast (Table 1). However, the characteristic pH optimum of pH 6 . 2 - 6 . 4 is retained (Fig. 3). A secondary inflection in the pH curve which occurs at pH 6.6 to 7.2 was ob-

Table 1. Endo-l,3-1,4-#-glucanase activity in yeast and bacteria Strain

X4003-5B (pJDB207)

X4003-5 B (pEHB9)

X4003-5 B (pEHB9-)

NCIB 8565

HB101 (pEHB3)

Specific activity a

0

2.34

0

2,101

111

a Endo-l,3-1,4-fl-glucanase activity was determined in crude cell extracts of yeast and culture supernatants of bacteria. Specific activity is expressed in n moles of reducing sugar released/min/mg protein, assayed at 40 °C and pH 6.4

474

E. Hinchliffe and W. G. Box: Expression of a prokaryotic gene in yeast

-13

-12 E

= "11 .~

-10

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"09

"08

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~1 "05

N .04

=,-,=

• 03 -02-

.01 I

3

I

t,

I

I

5

6

I

7

pH Fig. 3. The variation in endo-l,3-1,4-#-glucanase activity with pH was determined by measuring the release of reducing sugar from barley/3-gluean. Bacterial enzymes isolated from culture supernatants were incubated (0.05 ml in 1 ml reaction volume) at 40 °C for 10 min (B. subtilis o, HB101 (pEHB3) o), whereas crude cell extracts of X4003-5B (pEHB9) ([]) were incubated (0.2 ml in 1 ml reaction volume) at 40 °C for 60 rain

served in all three enzyme preparations, and would appear to be characteristic o f the B. subtilis enzyme (Moscatelli et al. 1961).

therefore, conclude that the endo-l,3-1,4-/3-glucanase gene orB. subtilis is expressed in S. cerevisiae to produce an active enzyme, which is clearly tolerated b y the host cell. There have been a number or reports concerning the expression o f prokaryotic genes in yeast, m a n y o f which have been o f plasrnid origin (Hollenberg 1979, 1981; Cohen et al. 1980; Jiminez and Davies 1980) although the chromosomal lacZ and ompA genes o f E. coli are also expressed (Panthier et al. 1980; Janowicz et al. 1982). Here we have demonstrated the expression o f a prokaryotic chromosomal gene from the Gram-positive bacterium B. subtilis. This gene appears to be transcribed and translated in a qualitatively correct manner, producing a functional enzyme with enzymic properties characteristic of those found in prokaryotic hosts. The fact that no extracellular endo-l,3-1,4-/3-glucanase activity could be detected in cultures ofS. cerevisiae harbouring pEHB9 is interesting and m a y be indicative o f the inability o f yeast to process the protein so as to promote secretion. Alternatively, difficulties in detecting the • enzyme in culture supernatants might be anticipated if the enzyme is predominantly periplasmic in cellular location since the activity will appear to be cell-associated. A more detailed examination o f the cellular location und secretory process will be greatly facilitated by the increased expression o f the gene in yeast. It will be necessary to increase gene expression and promote enzyme secretion prior to the introduction o f the gene into brewing yeast for successful industrial application.

Acknowledgements. We would like to thank the Directors of Bass PLC for permission to publish this paper. We would also like to thank C. W. Bamforth and E. F. Walton for many helpful discussions during the course of this work.

Discussion

References

The endo-l,3-1,4-~-glucanase gene o f B. subtilis strain NCIB 8565 has been located on a 1.4 kb PvuI-ClaI DNA fragment, contained within a 3.5 kbEcoRI DNA insert. A similar location has been assigned to an endo-1,3-1,4-/3-glucanase gene isolated from B. subtilis, strain NCIB 2117 (Cantwell and McConnel 1983). We have used the information obtained regarding the molecular location of the gene to construct a h y b r i d plasmid, pEHB9, which carries the LEU2 gene o f S. cerevisiae and the /3-glucanase gene o f B. subtilis and is capable o f autonomous replication in b o t h E. coli and S. cerevisiae. Plasmid transformants o f S. cerevisiae harbouring pEHB9 were found to display endo-l,3-1,4-~-glucanase activity, which could not be detected in transformants harbouting the parental plasmid pJDB207 or in a spontaneous leu-2 segregant o f X4003-5B (pEHB9). We,

Abd-E1-A1ATH, Phaff HJ (1968) Biochem J 109:347-360 Beggs JD (1978) Nature 275:104-109 Beggs JD (1981) Multiple-copy yeast plasmid vectors. In: yon Wettstein D, Stenderup A, Kielland-Brandt M, Friis J (eds) Molecular genetics in yeast. Alfred Benzon Symp 16 Munksgaard, Copenhagen, p 383 Boyer HW, Roulland-Dussoix D (1969) J Mol Bio141:459-472 Cantwell BA, McConnell DJ (1983) Gene 23:211-219 Chevallier MR, Aigle M (1979) FEBS Lett 108:179-180 Clewell DB~ Helinski DR (1969) Proc Natl Acad Sci USA 62: 1159-1166 Cohen JD, EccleshaU TR, Needleman RB, Federoff H, Buchferer B, Marmur J (1980) Proc Natl Acad Sci USA 77:1078-1082 Enkenlund J (1972) Proc Biochem August:27-29 Eyben D, Hupe J (1981) J Proc Eur Brew Conv. Barley and Malt Symp. Helsinki Monograph VI:201-212 Gjertsen P (1966) Proc Am Soc Brew Chem 113-120 Helling RB, Goodman HB, Boyer HW (1974) J Viro114:12351244

E. Hinchliffe and W. G. Box: Expression of a prokaryotic gene in yeast Hinchliffe E (1984) J Gen Microbiol 130:1285-1291 HoUenberg CP (1979) The expression of bacterial antibiotic resistance genes in the yeast Saccharomyces cerevisia. In: Timmis KN, Puhler A (eds) Plasmids of medical environmental and commercial importance. Elsevier, Amsterdam, p 148 Hollenberg CP (1981) Curr Top Microbiol Immunol 9 6 : 1 1 9 - 1 4 4 Janowicz ZA, Henning U, HoUenberg CP (1982) Gene 2 0 : 3 4 7 358 Jiminez A, Davies J (1980) Nature 287:869-871 Johnston GC, Pringle JR, Hartwell LH (1977) Exp Cell Res 105:79 -98 Kalb VF, Bernlohr RW (1977) Anal Biochem 82:362-371 Leedham PA, Savage DJ0 Crabb D~ Morgan GT (1975) Proc Eur Brew Conv, 15th Cong, Nice, p 201 Letters R (1969) J Inst Brew 7 5 : 5 4 - 6 0

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Letters R (1977) Proc Eur Brew Cony. 16th Cong, Amsterdam, p 211 Moscatelli EA, Ham EA, Rickes EL (1961) J Biol Chem 11: 2858-2862 Panthier JJ, Fourniet P, Heslot H, Rambach A (1980) Curt Genet 2:109.-113 Wilson GG, Murray NE (1979) Mol Gen Genet 1 5 6 : 2 0 3 - 2 1 4 Zahn G, Tippe-Schindler R, Messer W (1977) Mol Gen Genet 153:45--49

C o m m u n i c a t e d b y B. S. C o x Received March 24, 1984

Expression of the cloned endo-1,3-1,4-β-glucanase gene of Bacillus subtilis in Saccharomyces cerevisiae.

A cloned endo-1,3-1,4-β-glucanase gene from the Gram-positive bacterium B. subtilis has been located by deletion analysis on a 1.4 kb PvuI-ClaI DNA fr...
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