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Over-expression of the Saccharomyces cerevisiae exo-β-1,3-glucanase gene together with the Bacillus subtilis endo-β-1,3-1,4-glucanase gene and the Butyrivibrio fibrisolvens endo-β-1,4-glucanase gene in yeast
Journal of Biotechnology 55 (1997) 43 – 53
Over-expression of the Saccharomyces cere6isiae exo-b-1,3-glucanase gene together with the Bacillus subtilis endo-b-1,3-1,4-glucanase gene and the Butyri6ibrio fibrisol6ens endo-b-1,4-glucanase gene in yeast Pierre van Rensburg, Willem H. van Zyl, Isak S. Pretorius * Institute for Wine Biotechnology and Department of Microbiology, Uni6ersity of Stellenbosch, Stellenbosch 7600, South Africa Received 14 October 1996; received in revised form 18 March 1997; accepted 24 March 1997
Abstract The EXG1 gene encoding the main Saccharomyces cere6isiae exo-b-1,3-glucanase was cloned and over-expressed in yeast. The Bacillus subtilis endo-1,3-1,4-b-glucanase gene (beg1 ) and the Butyri6ibrio fibrisol6ens endo-b-1,4-glucanase gene (end1 ) were fused to the secretion signal sequence of the yeast mating pheromone a-factor (MFa1S ) and inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2P ) and terminator (ADH2T ). Constructs ADH2P MFa1S -beg1 -ADH2T and ADH2P -MFa1S -end1 -ADH2T, designated BEG1 and END1, respectively, were expressed separately and jointly with EXG1 in S. cere6isiae. The construction of fur1 ura3 S. cere6isiae strains allowed for the autoselection of these multicopy URA3 -based plasmids in rich medium. Enzyme assays confirmed that co-expression of EXG1, BEG1 and END1 enhanced glucan degradation by S. cere6isiae. © 1997 Elsevier Science B.V. Keywords: Glucanase; Over-expression; Bacillus subtilis; Butyri6ibrio fibrisol6ens; Saccharomyces cere6isiae
1. Introduction Polysaccharides play an important role in the production and processing of alcoholic beverages. Wines made from botrytized grapes often present
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serious clarification and filtration problems. Difficulties arise because of the presence of a high molecular weight polysaccharide, b-1,3-1,6-glucan, secreted by the grey mould Botrytis cinerea (Dubourdieu et al., 1981). This glucose polymer is released into the grape juice and later found in the wine. Glucan prevents the natural sedimentation of cloud particles in the grape must and causes filter stoppages. This negative effect can be overcome by using fining agents such as bentonite, or
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by centrifugation. Such treatment will force the sedimentation of the cloud but will not remove the glucan and filtration problems remain. Alcohol induces polymerization of the glucan molecules, thus more severe problems occur at the end of alcohol fermentation. Similarly, glucans in beer can also cause filter stoppages during the filtration process and a high glucan content may also lead to the formation of precipitates in the finished beer (Thomsen et al., 1988). Glucans in barley and cereal consist of short stretches of b-1,4-linked glucose moieties (cellotriosyl and cellotraosyl) interrupted by single b-1,3-linkages (Bielecki and Galas, 1991). The mixed glycosidic linkages present in b-glucans render them more soluble than the major nonstructural polysaccharide cellulose, which consists only of long b-1,4-linked glucose stretches. Furthermore, barley glucans can be antinutritional to animals and the glucans in cereal grain-based diets have deleterious side-effects, such as sticky droppings (Walsh et al., 1993). Although various glucanase activities have been described for Saccharomyces cere6isiae (Farkas et al., 1973), wine and brewers’ yeasts do not secrete enzymes that can degrade glucans to fermentable sugars. This is not surprising since most strains of S. cere6isiae produce a cell wall-bound endo-b1,3-glucanase (Mrsa et al., 1993) and extracellular exo-b-1,3-glucanases (Nebreda et al., 1986; Kuranda and Robbins, 1987) with b-1,3- and b-1,6-activities (Nombela et al., 1988), but no glucanase with b-1,4-activity. It will therefore be advantageous to introduce glucanase-encoding genes into S. cere6isiae strains used in the wine and brewing industries. One can also manipulate probiotic yeast strains to secrete b-glucanases and incorporate them into barley-containing animal feeds to alleviate some of the adverse effects on animal digestion. To this end, our laboratory has expressed the endo-b-1,4-glucanase gene from Butyri6ibrio fibrisol6ens (Van Rensburg et al., 1994), the cellodextrinase gene from Ruminococcus fla6efaciens (Van Rensburg et al., 1995) and the cellobiohydrolase gene from Phanerochaete chrysosporium (Van Rensburg et al., 1996) in S. cere6isiae.
The hydrolysis of glucans occurring in grape must, beer wort and probiotic animal feed requires the synergistic action of different types of glucanases. Therefore, to create a recombinant S. cere6isiae strain that can degrade these glucans, our objective was to construct a yeast expression cassette consisting of genes encoding the three main classes of glucanases. Bacillus subtilis secretes an industrially useful b-1,3-1,4-glucanase that specifically hydrolyzes barley glucan and lichenan (Cantwell and McConnell, 1983). Unlike the B. fibrisol6ens endo-b-1,4-glucanase that hydrolyzes internal b-1,4-linkages next to b-1,4linked glucose residues, and the S. cere6isiae exo-b-1,3-glucanases that hydrolyze b-1,3-bonds next to b-1,3-linked glucose residues and b-1,6 linkages, the B. subtilis endo-b-1,3-1,4-glucanase only hydrolyzes b-1,4-linkages adjacent to b-1,3linkages (Wolf et al., 1995). Here we describe the complementation of the S. cere6isiae glucanase activities by over-expressing the S. cere6isiae exob-1,3-glucanase gene (EXG1 ) together with the B. subtilis endo-b-1,3-1,4-glucanase gene (beg1 ) and the B. fibrisol6ens endo-b-1,4-glucanase gene (end1 ) in S. cere6isiae.
2. Materials and methods
2.1. Microbial strains and plasmids The sources and relevant genotypes of bacterial and yeast strains, as well as plasmids used in this study, are listed in Table 1. Plasmid pEHB9, carrying the cloned endo-b-1,3-1,4-glucanase gene (beg1 ) from B. subtilis NCIB 8565, was obtained from Dr E. Hinchliffe (Hinchliffe, 1984; Hinchliffe and Box, 1984), whereas the endo-b-1,4-glucanase gene (end1 ) from B. fibrisol6ens H17c was kindly provided by Dr D.R. Woods (Berger et al., 1989).
2.2. Media and screening procedures Bacteria were grown in Luria-Bertani broth (LB; Sambrook et al., 1989). Escherichia coli transformants were grown in LB supplemented with ampicillin (100 mg ml − 1). YPD medium
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Table 1 Microbial strains and plasmids used in the present study Strain or plasmid Yeast strains: Y294 Escherichia coli strains: E. coli DH5a Plasmids: YEp352 pEHB9 pPK4 pPK5 pBG1 pEX191 pEXG pJC1 pDLG4 pAR1 pBEX pEND pBEG pBEE pPE pDF1 a
Relevant genotypea
Source or reference
a leu2 -3, 112 ura3 -52 his3 trp1 -289
This laboratory
supE44 DlacU169 (ø80lacZDM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1
Sambrook et al. (1989)
ApR URA3 ApR beg1 ApR beg1 ApR beg1 ApR URA3 EXG1 ApR EXG1 ApR URA3 EXG1 ApR URA3 PGK1PT ApR URA3 ADH2P -MFa1S -ADH2T ApR URA3 ADC1P -MFa1S -end1 ApR URA3 ADH2P -MFa1S -beg1 -ADH2T EXG1 ApR URA3 ADH2P -MFa1S -end1 -ADH2T ApR URA3 ADH2P -MFa1S -beg1 -ADH2T ApR URA3 ADH2P -MFa1S -end1 -ADH2T ADH2P -MFa1S -beg1 -ADH2T EXG1 ApR TRP1 FUR1 ApR fur1::LEU2
Hill et al. (1986) Hinchliffe (1984) This work This work This work This work This work Crous et al. (1995) La Grange et al. (1996) Van Rensburg et al. (1995) This work This work This work This work Kern et al. (1990) La Grange et al. (1996)
The constructs ADH2P -MFa1S -beg1 -ADH2T and ADH2P -MFa1S -end1 -ADH2T were designated BEG1 and END1, respectively.
(containing 1% yeast extract, 2% peptone and 2% glucose) and YPGE medium (containing 1% yeast extract, 2% peptone, 3% glycerol and 2% ethanol) were used to grow yeast cells. A laboratory strain of S. cere6isiae (Y294) was transformed with the recombinant plasmids and the transformants were grown in SC-Ura (containing 0.67% yeast nitrogen base without amino acids, 2% glucose and all required growth factors except uracil). Solid media contained 2% agar, unless otherwise indicated. Bacteria and yeasts were routinely cultured at 37°C and 30°C, respectively. Glucanase-producing yeast transformants were identified on YPD plates containing 0.3% medium viscosity carboxymethylcellulose (CMC; Sigma PC4888; Sigma, St. Louis, Mo., USA), 0.1% barley b-glucan (Sigma) or 0.4% lichenan (Sigma). The yeast transformants were grown for 2 days and the colonies were rinsed off the plates with TE buffer (10 mM Tris. HCl, 1 mM EDTA, pH 7) before staining the plates with 0.1% Congo red,
followed by destaining with 1 M NaCl in the case of carboxymethylcellulose (Teather and Wood, 1982). Colonies showing glucanase activity were identified by the formation of a clear zone around the colony.
2.3. Recombinant DNA and genetic techniques Standard methods were used for the isolation of plasmid DNA, genomic DNA and RNA, subcloning and manipulation of DNA fragments, and transformation of E. coli (Ausubel et al., 1987; Sambrook et al., 1989). Restriction endonucleases, T4 DNA ligase, Klenow (large) fragment of E. coli DNA polymerase I, DNase I and calf intestinal alkaline phosphatase were purchased from Boehringer Mannheim (Randburg, RSA) and used as recommended by the supplier. Nucleotide sequencing performed by the dideoxy chain-terminating method (Sanger et al., 1977) for double-stranded plasmid templates using a Seque-
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nase kit (United States Biochemical Corporation, Cleveland, Oh., USA). The [35S]-dATP used for the sequencing reactions was purchased from Amersham International (Buckinghamshire, UK). DNA amplification was performed by the polymerase chain reaction (PCR) technique (Innis and Gelfand, 1990; Saiki et al., 1988). Premixed PCR reagents (GeneAmp PCR reagent kit with Ampli Taq DNA polymerase) were purchased from Perkin-Elmer/Cetus (Norwalk, Ct., USA) and used according to the supplier’s recommendations. The following single-stranded oligodeoxyribonucleotides (restriction sites are underlined) were synthesized by the phosphoramidate method on a AutogenTM 650 DNA synthesizer: PBEG-5% (HindIII restriction site) 5%-GCCGAAGCTTTGCAAACAGGCGGATCGTTT-3% PBEG-3% (HindIII and NotI restriction sites) 5%-ACGTAAGCTTGCGGCCGCCATTATTTTTTTGTATAGCGCA-3%
2.4. Construction of a genomic library of S. cere6isiae Total genomic DNA from S. cere6isiae carrying the EXG1 exo-b-1,3-glucanase gene was partially digested with Sau3A. DNA fragments larger than 5 kb were recovered and inserted by ligation into the BamHI site of the E. coli-S. cere6isiae shuttle plasmid YEp24. A genomic library of 25 000 ApR (ampicillin resistant) clones, 90% of which were tetracycline sensitive (TcS), was obtained. Recombinant plasmid DNA prepared from the library was used to transform S. cere6isiae Y294 to Ura+. The Ura + transformants were screened for the EXG1 -encoded exo-b-1,3-glucanase activity by spraying SC-Ura plates with a solution containing 0.04% (w/v) 4-methyl-umbelliferyl-b-D-glucoside (MUG) in 50 mM acetate buffer (pH 5.2). After incubation at 37°C for 30 min, the plates were examined under UV light (254 nm). Transformants in which the exo-b-1,3-glucanase was overexpressed, appeared fluorescent because of the hydrolysis of MUG. A YEp24-based plasmid, pBG1, that conferred enhanced exo-b-1,3-glucanase activity on S. cere6isiae Y294, was recovered from one of the transformants and characterized by restriction analysis.
2.5. Southern and Northern blot hybridization Genomic DNA was isolated from S. cere6isiae, digested with NsiI and NcoI, separated on a 1% agarose gel and transferred to a nitrocellulose Hybond-N membrane (Amersham). Southern and Northern blot hybridizations were carried out as described by Sambrook et al. (1989). The presence of BEG1, END1 and EXG1 mRNA was confirmed by probing with an internal 720-bp HindIII DNA fragment of the beg1 gene, a 1563-bp HindIII DNA fragment of the B. fibrisol6ens end1 gene, and a 764-bp XbaI-BglII DNA fragment of the EXG1 gene, respectively. The DNA probes were labeled using a random primed DNA labeling kit (Boehringer Mannheim) and [a-32P]dATP (Amersham).
2.6. Yeast transformation S. cere6isiae Y294 was transformed with episomal-derived plasmid DNA by the lithium-acetate method of Gietz and Schiestl (1991). Plasmids pBEG, pEND and pBEE were maintained as autonomous circular minichromosomes in the yeast cells. The presence of these plasmids in the recombinant yeasts was verified by recovering them from the yeast cells, re-introducing them into E. coli and confirming their restriction endonuclease maps with those of the original plasmids.
2.7. Enzyme assays S. cere6isiae Y294, transformed separately with plasmid YEp352 and recombinant plasmids pBEG, pEND and pBEE, was cultured in 100 ml YPGE medium at 30°C. Samples were collected at different time points after inoculation and the same amount of cells was harvested from each of the samples. The culture supernatants, containing the secreted glucanase, were assayed with barley b-glucan (20 mg ml-1; Sigma) as substrate at 50°C. Appropriate dilutions of the supernatant were done using 0.05 M citrate-phosphate buffer (pH 6.2). For every 900 ml substrate 100 ml tant was added. Aliquots (1 ml) were removed in duplicate at intervals, heated (100°C) for 15 min
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and cooled to room temperature. Subsequently, 200 ml Congo red (100 mg ml-1) was added and the mixture diluted with buffer to 2 ml. Absorbancy was measured at 540 nm (Wood et al., 1988).
3. Results
3.1. Characterization of BEG1 Overlapping fragments of beg1 were subcloned from pPK5 into pUC18. The DNA sequence of the beg1 gene was determined by sequencing both strands. The nucleotide sequence of the 1.4-kb P6uI-ClaI fragment contained a single open reading frame (ORF) of 720 bp, encoding 240 amino acid residues (GenBank accession number U60830). The promoter region was located upstream of the ORF with a -35 sequence of TTCGAC followed by a 19-bp space and a -10 region of GATCAT. The deduced amino acid sequence from the B. subtilis NCIB 8565 beg1 -encoded protein (Beg1 ) was compared with those of endo-1,3-1,4-glucanases from five Bacillus species: Beg1 showed higher homology with BglA of B. amyloliquefaciens (97%; Hofemeister et al., 1986) than with BglS of B. subtilis (91%; Murphy et al., 1984). The homologies with BglL of B. licheniformis (Lloberas et al., 1991), GluM of B. marcerans (Borriss et al., 1990) and GluB of B. polymyxa (Gosalbes et al., 1991) were 84%, 76% and 75%, respectively (Fig. 1). The protein homology data suggest that Beg1 of B. subtilis NCIB 8565 and BglA of B. amyloliquefaciens are closely related even with regard to their leader peptide sequences. BglS of B. subtilis and BglL of B. licheniformis are more distantly related to Beg1 and BglA. However, GluM of B. marcerans and GluB of B. polymyxa differ more substantially from the other four endo-b-1,3-1,4-glucanases, particularly within their leader peptide sequences. These data correspond well with the morphological grouping of Bacillus species into three groups with B. amyloliquefaciens, B. subtilis and B. licheniformis within Group I and B. marcerans and B. polymyxa within Group II (Slepecky, 1992).
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3.2. Construction of BEG1, END1 and EXG1 A 3.5-kb EcoRI fragment from plasmid pEHB9 (Hinchliffe and Box, 1984), carrying the endo-b1,3-1,4-glucanase gene (beg1 ), was subcloned into pUC18, generating plasmid pPK4. The beg1 gene has been located on a 1.4-kb P6uI-ClaI fragment, contained within the 3.5-kb EcoRI DNA insert. The 1.4-kb P6uI-ClaI fragment from plasmid pPK4 was isolated, end-filled with Klenow enzyme and subcloned into the unique SmaI site of pUC18, generating recombinant plasmid pPK5. Based on the nucleotide sequence of beg1, oligodeoxynucleotides PBEG-5% and PBEG-3% were synthesized and used as primers to amplify a 720-bp HindIII fragment containing the beg1 gene. The resulting PCR product was subcloned into the HindIII site of the multicopy E. coli – S. cere6isiae shuttle plasmid pDLG4 (La Grange et al., 1996), thereby generating plasmid pBEG. In pBEG, the beg1 ORF was fused in-frame to the secretion signal sequence of the yeast mating pheromone a-factor (MFa1S ) and inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2P ) and terminator (ADH2T ). The ADH2P -MFa1S -beg1 -ADH2T construct was designated BEG1. Previously, the B. fibrisol6ens endo-b-1,4-glucanase gene (end1 ) was also fused in-frame to ADH2P -MFa1S and linked to ADH2T. Construct ADH2P -MFa1S -end1 -ADH2T (designated END1 ) was subcloned into the multicopy E. coli-S. cere6isiae shuttle plasmid, YEp352, resulting in plasmid pEND1 (Van Rensburg et al., 1996). A 3.95-kb BamHI-NheI fragment from plasmid pBG1 (YEp24 containing the EXG1 gene from S. cere6isiae) was subcloned into the BamHI and XbaI sites of pUC19, thereby generating plasmid pEX191. A 3.5-kb HindIII fragment was subcloned from pEX191 into the HindIII restriction site of YEp352, resulting in pEXG. This multicopy E. coli-S. cere6isiae shuttle plasmid contains the EXG1 gene with its native promoter and terminator sequences. A partial digestion of pBEG with BamHI produced a 2857-bp fragment containing theBEG1 construct. This fragment was cloned into the
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Fig. 1. The level of homology among the deduced amino acid sequences of endo-b-1,3-1,4-glucanases of the following Bacillus species: B. subtilis strain NCIB 2117, B. amyloliquefaciens (BglA), B. licheniformis (BglL), B. macerans (GluM) and B. polymyxa (GluB).
BamHI site of pEXG, generating plasmid pBEX. A 6357-bp P6uII DNA fragment containing the BEG1 and EXG1 constructs was subcloned from
pBEX into the unique SmaI site of pEND (Van Rensburg et al., 1996), thereby generating pBEE (Fig. 2).
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Fig. 2. Restriction maps of recombinant plasmids pBEG, pEND and pBEE. All three plasmids are multi-copy E. coli-S. cere6isiae shuttle vectors. The bacterial structural genes were inserted into expression-secretion cassettes (ADH2P -MFa1S and ADH1T). Plasmid pBEG (BEG1 ) contains the B. subtilis beg1 endo-b-1,3-1,4-glucanase gene, plasmid pEND (END1 ) contains the B. fibrisol6ens end1 endo-b-1,4-glucanase gene and plasmid pBEE (BEG1, END1 and EXG1 ) contains the end1 gene together with the beg1 and EXG1 genes. EXG1 encodes the main exo-b-1,3-glucanase in S. cere6isiae. Constructs ADH2P -MFa1S -beg1 -ADH2T and ADH2P -MFa1S -end1 -ADH2T ) were designated BEG1 and END1, respectively.
3.3. Expression of BEG1, END1 and EXG1 in autoselecti6e strains of S. cere6isiae Plasmids YEp352, pBEG (BEG1 ), pEND (END1 ) and pBEE (BEG1, END1 and EXG1 ) were transformed into S. cere6isiae Y294. Autoselective strains that would allow plasmid selection in uracil-containing medium were constructed according to the method of Loison et al. (1986). The wild-type FUR1 gene of S. cere6isiae strains Y294[YEp352], Y294[BEG1 ], Y294[END1 ] and Y-
294 [BEG1, END1 and EXG1 ] was replaced with a defective fur1 ::LEU2 allele by homologous recombination (Rothstein, 1983), using the 3.27-kb NcoI/NsiI linear DNA fragment from plasmid pDF1 (La Grange et al., 1996). The FUR1 gene from S. cere6isiae encodes uracil phosphoribosyltransferase which catalyzes the conversion of uracil into uridine 5%-phosphate in the pyrimidine salvage pathway (Kern et al., 1990). By disrupting this gene, fur1::LEU2 strains of S. cere6isiae Y294 were not able to utilize uracil from the culture medium and were therefore not viable, unless they
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Fig. 3. Southern blot analysis of fur1::LEU2 disruption in the autoselective S. cere6isiae Y294 transformants. Total DNA isolated from S. cere6isiae Y294 (lane 1 ), Y294[ fur1::LEU2 BEG1 ] (lane 2 ), Y294[ fur1::LEU2 END1 ] (lane3 ), and Y294[ fur1::LEU2 BEG1,END1 and EXG1 ] (lane 4 ) was digested with NcoI and NsiI, and probed with a [a-32P]-dATPlabeled NcoI/NsiI fragment of the FUR1 gene, derived from plasmid pPE (Kern et al. 1990).
possessed a URA3 -containing plasmid to synthesize uridine 5%-phosphate de novo. Leu + yeast transformants were selected on SC-Leu-Ura plates. The DNA-DNA hybridization by Southern blot analysis confirmed the integration of the defective fur1::LEU2 allele into the FUR1 gene (Fig. 3). The DNA-RNA hybridization by Northern blot analysis revealed the presence of the BEG1, END1 and EXG1 transcripts in S. cere6isiae Y294 containing pBEE (Fig. 4), the BEG1 transcript in Y294 carrying pBEG, and END1 transcript in
Fig. 5. Time course of extracellular glucanase activity produced by yeast transformants. The activity of the transformant Y294[ fur1::LEU2 BEG1 ] is depicted by (), Y294[ fur1::LEU2 END1 ] is depicted by (), Y294[ fur1::LEU2 BEG1 END1 EXG1 ] is depicted by ( ).
Y294 carrying pEND. The extracellular glucanase activities produced by the various S. cere6isiae Y294 transformants are shown in Fig. 5.
4. Discussion The results presented here describe the construction of autoselective, glucanolytic strains of S. cere6isiae by complementing the endogenous exo-b-1,3-glucanase and the cell-wall-bound endo-b-1,3-glucanase activities of the yeast with
Fig. 4. Northern blot analysis of BEG1 (A) END1 (B) and EXG1 (C) transcripts prepared from yeast cells. RNA samples were loaded as follows: (A) lane 1, Y294[YEp352]; lane 2, Y294[pBEG]; lane 3, Y294[pBEE], and (B) lane 1, Y294[YEp352]; lane 2, Y294[pEND]; lane 3, Y294[pBEE]; and (C) lane 1, Y294[YEp352]; lane 2, Y294[pBEE]. The nylon membrane was probed with internal fragments of the B. subtilis beg1, B. fibrisol6ens end1 and S. cere6isiae EXG1 genes. A RNA was used as a molecular size standard and the sizes (in kb) are indicated on the left.
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the B. subtilis endo-b-1,3-1,4-glucanase gene (beg1 ), the B. fibrisol6ens endo-b-1,4-glucanase gene (end1 ), and the over-expression of the S. cere6isiae exo-b-1,3-glucanase gene (EXG1 ). Transcription of both the BEG1 and END1 constructs was directed by the ADH2 promoter and termination sequences. The transcription of the EXG1 gene was directed by its native promoter and termination sequences. DNA-RNA hybridization by Northern blot analysis revealed the presence of the BEG1 transcript in S. cere6isiae transformant Y294[BEG1 ] carrying pBEG, the END1 transcript in Y294[END1 ] containing pEND, and the BEG1, END1 and EXG1 transcripts in Y294[BEG1 +END1 +EXG1 ] carrying pBEE (Fig. 1). The Northern blot in Fig. 4B clearly showed that transcription levels in the transformants containing plasmid pBEE were much lower than in the transformants containing pBEG or pEND. This is possibly caused by the titration of transcription factors or that the large size of pBEE caused the copy number of the episomal plasmid to decrease. Efficient secretion of the endo-b-1,4-glucanase and endo-b-1,3-1,4-glucanase from S. cere6isiae transformants was facilitated by in-frame fusions of the coding sequence for these mature enzymes to the MFa1 -encoded leader sequence. The 89amino acid MFa1 signal peptide, ending in the Lys-Arg-(Glu-Ala)2 hexapeptide (Kurjan and Herskowitz, 1982), has been used extensively for the secretion of various heterologous proteins by S. cere6isiae (Bitter et al., 1984, 1989; Brake, 1990). The native secretion signal sequence of the exo-1,3-glucanase mediated secretion in the S. cere6isiae transformants. The Northern blot analysis (Fig. 4) and results from time-course experiments (Fig. 5) indicated that not only was the transcription of the BEG1, END1 and EXG1 constructs efficiently promoted, but that the S. cere6isiae transformants also secreted biologically active endo-b-1,3-1,4-glucanase, endo-b-1,4-glucanase and/or exo-b-1,3-glucanase. It is relatively difficult to assay for endo-b-1,31,4-glucanase activity by measuring the amount of reducing sugars. The problem arises due to the fact that in barley b-glucan there are only a few 1,4-linkages next to 1,3-linkages, allowing for only
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a few places where the enzyme can cut the glucan chain. This results in the production of only a few reducing sugar ends and if measured by the dinotrosalicyclic acid (DNS) method (Miller et al., 1960) an effect is not detected. Interestingly, although only a few reducing sugar ends are formed, a large clearing zone is formed during the plate assay with Congo red. Results of the plate assays we performed clearly showed that the endo-b-1,3-1,4-glucanase only utilized barley bglucan and lichenan, but not CMC (results not shown), whereas the endo-b-1,4-glucanase hydrolyzed all three substrates. These results confirmed that endo-b-1,3-1,4-glucanases only hydrolyze b-1,4-linkages adjacent to b-1,3-linkages (Wolf et al., 1995), because CMC contains no 1,4-linkages next to 1,3-linkages. Based on the above results, we decided to use a liquid assay utilizing the breakdown of the glucan/Congo red complex (Wood et al., 1988). The data indicated that transformants containing plasmid pBEG (BEG1 ) generated a maximum of 16% breakdown of the glucan/Congo red complex in 10 h, whereas the transformants containing plasmid pEND (END1 ) produced 34% breakdown of the complex. Transformants carrying pBEE (BEG1, END1 and EXG1 ) produced a maximum breakdown of 37%. This clearly indicated that co-expression did not significantly improve the breakdown of the barley-b-glucan/Congo red complex in this specific time span, but the results did not show the size and type of the end products. Significantly different results could possibly have been obtained if a different type of glucan had been used which contains more 1,3-linkages or even 1,6-linkages (glucan produced by B. cinerea), since the exo-b-1,3-glucanase also contains 1,6-activity.
Acknowledgements The authors thank Drs E. Hinchliffe (Bass PLC, Burton-on-Trent) and D.R. Woods (University of Cape Town) for providing the B. subtilis endo-b-1,3-1,4-glucanase and B. fibrisol6ens endob-1,4-glucanase genes, respectively. We gratefully acknowledge the financial support from the Foun-
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dation for Research Development, Industrial Development Corporation of Southern Africa and the South African wine industry (Winetech).
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Hinchliffe, E., 1984. Cloning and expression of a B. subtilis endo-1,3-1,4-b-D-glucanase gene in E. coli K12. J. Gen. Microbiol. 130, 1285 – 1291. Hinchliffe, E., Box, W.G., 1984. Expression of the cloned endo-1,3-1,4-b-D-glucanase gene of B. subtilis in S. cere6isiae. Curr. Genet. 8, 471 – 475. Hofemeister, J., Kurtz, A., Borriss, R., Knowles, J., 1986. The b-glucanase from Bacillus amyloliquefaciens shows extensive homology with that of Bacillus subtilis. Gene 49, 177 – 187. Innis, M.A., Gelfand, D.H., 1990. Optimizing of PCRs. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR protocols. Academic Press, San Diego, Calif., pp. 3 – 12. Kern, L., De Montigny, J., Jund, R., Lacroute, F., 1990. The FUR1 gene of Saccharomyces cere6isiae: cloning, structure and expression of wild-type and mutant alleles. Gene 88, 149 – 157. Kuranda, M.J., Robbins, P., 1987. Cloning and heterologous expression of glycosidase genes from Saccharomyces cere6isiae. Proc. Natl. Acad. Sci. USA 84, 2585 – 2589. Kurjan, J., Herskowitz, I., 1982. Structure of a yeast pheromone gene (MFa): a putative a-factor precursor contains four tandem copies of mature a-factor. Cell 30, 933 – 943. La Grange, D.C., Pretorius, I.S., Van Zyl, W.H., 1996. Expression of a Trichoderma reesei b-xylanase gene (XYN2 ) in Saccharomyces cere6isiae. Appl. Environ. Microbiol. 62, 1036 – 1044. Lloberas, J., Perez-Pons, J.A., Querol, E., 1991. Molecular cloning, expression and nucleotide sequence of the endo-b1,3-1,4-D-glucanase gene from Bacillus licheniformis. Eur. J. Biochem. 197, 337 – 343. Loison, G., Nguyen-Juilleret, M., Alouani, S., Marquet, M., 1986. Plasmid-transformed URA3 FUR1 double-mutants of S. cere6isiae: an autoselection system applicable to the production of foreign proteins. Biotechnol. 4, 433 – 437. Miller, G.L., Blum, R., Glennon, W.M., Burton, A.L., 1960. Measurement of carboxymethylcellulase activity. Anal. Biochem. 2, 127 – 132. Mrsa, V., Klebl, F., Tanner, W., 1993. Purification and characterization of the Saccharomyces cere6isiae BGL2 gene product, a cell wall endo-b-1,3-glucanase. J. Bacteriol. 175, 2102 – 2106. Murphy, N., McConnell, D.J., Cantwell, B.A., 1984. The DNA sequence of the gene and genetic control sites for the excreted B. subtilis enzyme b-glucanase. Nucleic Acids Res. 12, 5355 – 5367. Nebreda, A.R., Villa, T.G., Villanueva, J.R., del Rey, F., 1986. Cloning of genes related to exo-b-glucanase production in Saccharomyces cere6isiae: characterization of an exo-b-glucanase structural gene. Gene 47, 245 – 259. Nombela, C., Molina, M., Cenamor, R., Sanchez, M., 1988. Yeast b-glucanases: a complex system of secreted enzymes. Microbiol. Sci. 328 – 332. Rothstein, R.J., 1983. One-step gene disruption in yeast. Methods Enzymol. 101, 202 – 211.
P. 6an Rensburg et al. / Journal of Biotechnology 55 (1997) 43–53 Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., Ehrlich, H.A., 1988. Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 287–491. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463 – 5467. Slepecky, R.A., 1992. What is a Bacillus. In: Doi, R.H., McGloughlin, M. (Eds.), Biology of bacilli: applications to industry. Butterworth-Heinemann, Boston, pp. 1–21. Teather, R.M., Wood, P.J., 1982. Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 53, 41–46. Thomsen, K.K., Jackson, E.A., Brenner, K., 1988. Genetic engineering of yeast: construction of strains that degrade b-glucans with aid of a Barley gene. ASBC Journal 46, 31– 36. Van Rensburg, P., Van Zyl, W.H., Pretorius, I.S., 1994. Expression of the Butyri6ibrio fibrisol6ens endo-b-1,4-glucanase gene together with the Erwinia pectate lyase and
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polygalacturonase genes in Saccharomyces cere6isiae. Curr. Genet. 27, 17 – 22. Van Rensburg, P., Van Zyl, W.H., Pretorius, I.S., 1995. Expression of the Ruminococcus fla6efaciens cellodextrinase gene in Saccharomyces cere6isiae. Biotechnol. Lett. 17, 481 – 486. Van Rensburg, P., Van Zyl, W.H., Pretorius, I.S., 1996. Co-expressiom of a Phanerochaete chrysosporium cellobiohydrolase gene and a Butyri6ibrio fibrisol6ens endo-b-1,4glucanase gene in Saccharomyces cere6isiae. Curr. Genet. 30, 246 – 250. Walsh, G.A., Power, R.F., Headon, D.R., 1993. Enzymes in the animal-feed industry. Trends. Biotechnol. 11, 424 – 430 Wolf, M., Geczi, A., Simon, O., Borriss, R., 1995. Genes encoding xylan and b-glucan hydrolysing enzymes in Bacillus subtilis: characterization, mapping and construction of strain deficient in lichenase, cellulase and xylanase. Microbiol. 141, 281 – 290. Wood, P.J., Erfle, J.D., Teather, R.M., 1988. Use of complex formation between Congo Red and polysaccharides in detection and assay of polysaccharide hydrolases. In: Wood, W.A., Kellog, S.T. (Eds.), Methods in Enzymology. Biomass. Part A. and Hemicellulose. Academic Press, Inc., San Diego, pp. 3 – 11.
Over-expression of the Saccharomyces cere6isiae exo-b-1,3-glucanase gene together with the Bacillus subtilis endo-b-1,3-1,4-glucanase gene and the Butyri6ibrio fibrisol6ens endo-b-1,4-glucanase gene in yeast Pierre van Rensburg, Willem H. van Zyl, Isak S. Pretorius * Institute for Wine Biotechnology and Department of Microbiology, Uni6ersity of Stellenbosch, Stellenbosch 7600, South Africa Received 14 October 1996; received in revised form 18 March 1997; accepted 24 March 1997
Abstract The EXG1 gene encoding the main Saccharomyces cere6isiae exo-b-1,3-glucanase was cloned and over-expressed in yeast. The Bacillus subtilis endo-1,3-1,4-b-glucanase gene (beg1 ) and the Butyri6ibrio fibrisol6ens endo-b-1,4-glucanase gene (end1 ) were fused to the secretion signal sequence of the yeast mating pheromone a-factor (MFa1S ) and inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2P ) and terminator (ADH2T ). Constructs ADH2P MFa1S -beg1 -ADH2T and ADH2P -MFa1S -end1 -ADH2T, designated BEG1 and END1, respectively, were expressed separately and jointly with EXG1 in S. cere6isiae. The construction of fur1 ura3 S. cere6isiae strains allowed for the autoselection of these multicopy URA3 -based plasmids in rich medium. Enzyme assays confirmed that co-expression of EXG1, BEG1 and END1 enhanced glucan degradation by S. cere6isiae. © 1997 Elsevier Science B.V. Keywords: Glucanase; Over-expression; Bacillus subtilis; Butyri6ibrio fibrisol6ens; Saccharomyces cere6isiae
1. Introduction Polysaccharides play an important role in the production and processing of alcoholic beverages. Wines made from botrytized grapes often present
* Corresponding author. Tel: +27 21 8084730; fax: +27 21 8083771; e-mail: [email protected]
serious clarification and filtration problems. Difficulties arise because of the presence of a high molecular weight polysaccharide, b-1,3-1,6-glucan, secreted by the grey mould Botrytis cinerea (Dubourdieu et al., 1981). This glucose polymer is released into the grape juice and later found in the wine. Glucan prevents the natural sedimentation of cloud particles in the grape must and causes filter stoppages. This negative effect can be overcome by using fining agents such as bentonite, or
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by centrifugation. Such treatment will force the sedimentation of the cloud but will not remove the glucan and filtration problems remain. Alcohol induces polymerization of the glucan molecules, thus more severe problems occur at the end of alcohol fermentation. Similarly, glucans in beer can also cause filter stoppages during the filtration process and a high glucan content may also lead to the formation of precipitates in the finished beer (Thomsen et al., 1988). Glucans in barley and cereal consist of short stretches of b-1,4-linked glucose moieties (cellotriosyl and cellotraosyl) interrupted by single b-1,3-linkages (Bielecki and Galas, 1991). The mixed glycosidic linkages present in b-glucans render them more soluble than the major nonstructural polysaccharide cellulose, which consists only of long b-1,4-linked glucose stretches. Furthermore, barley glucans can be antinutritional to animals and the glucans in cereal grain-based diets have deleterious side-effects, such as sticky droppings (Walsh et al., 1993). Although various glucanase activities have been described for Saccharomyces cere6isiae (Farkas et al., 1973), wine and brewers’ yeasts do not secrete enzymes that can degrade glucans to fermentable sugars. This is not surprising since most strains of S. cere6isiae produce a cell wall-bound endo-b1,3-glucanase (Mrsa et al., 1993) and extracellular exo-b-1,3-glucanases (Nebreda et al., 1986; Kuranda and Robbins, 1987) with b-1,3- and b-1,6-activities (Nombela et al., 1988), but no glucanase with b-1,4-activity. It will therefore be advantageous to introduce glucanase-encoding genes into S. cere6isiae strains used in the wine and brewing industries. One can also manipulate probiotic yeast strains to secrete b-glucanases and incorporate them into barley-containing animal feeds to alleviate some of the adverse effects on animal digestion. To this end, our laboratory has expressed the endo-b-1,4-glucanase gene from Butyri6ibrio fibrisol6ens (Van Rensburg et al., 1994), the cellodextrinase gene from Ruminococcus fla6efaciens (Van Rensburg et al., 1995) and the cellobiohydrolase gene from Phanerochaete chrysosporium (Van Rensburg et al., 1996) in S. cere6isiae.
The hydrolysis of glucans occurring in grape must, beer wort and probiotic animal feed requires the synergistic action of different types of glucanases. Therefore, to create a recombinant S. cere6isiae strain that can degrade these glucans, our objective was to construct a yeast expression cassette consisting of genes encoding the three main classes of glucanases. Bacillus subtilis secretes an industrially useful b-1,3-1,4-glucanase that specifically hydrolyzes barley glucan and lichenan (Cantwell and McConnell, 1983). Unlike the B. fibrisol6ens endo-b-1,4-glucanase that hydrolyzes internal b-1,4-linkages next to b-1,4linked glucose residues, and the S. cere6isiae exo-b-1,3-glucanases that hydrolyze b-1,3-bonds next to b-1,3-linked glucose residues and b-1,6 linkages, the B. subtilis endo-b-1,3-1,4-glucanase only hydrolyzes b-1,4-linkages adjacent to b-1,3linkages (Wolf et al., 1995). Here we describe the complementation of the S. cere6isiae glucanase activities by over-expressing the S. cere6isiae exob-1,3-glucanase gene (EXG1 ) together with the B. subtilis endo-b-1,3-1,4-glucanase gene (beg1 ) and the B. fibrisol6ens endo-b-1,4-glucanase gene (end1 ) in S. cere6isiae.
2. Materials and methods
2.1. Microbial strains and plasmids The sources and relevant genotypes of bacterial and yeast strains, as well as plasmids used in this study, are listed in Table 1. Plasmid pEHB9, carrying the cloned endo-b-1,3-1,4-glucanase gene (beg1 ) from B. subtilis NCIB 8565, was obtained from Dr E. Hinchliffe (Hinchliffe, 1984; Hinchliffe and Box, 1984), whereas the endo-b-1,4-glucanase gene (end1 ) from B. fibrisol6ens H17c was kindly provided by Dr D.R. Woods (Berger et al., 1989).
2.2. Media and screening procedures Bacteria were grown in Luria-Bertani broth (LB; Sambrook et al., 1989). Escherichia coli transformants were grown in LB supplemented with ampicillin (100 mg ml − 1). YPD medium
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Table 1 Microbial strains and plasmids used in the present study Strain or plasmid Yeast strains: Y294 Escherichia coli strains: E. coli DH5a Plasmids: YEp352 pEHB9 pPK4 pPK5 pBG1 pEX191 pEXG pJC1 pDLG4 pAR1 pBEX pEND pBEG pBEE pPE pDF1 a
Relevant genotypea
Source or reference
a leu2 -3, 112 ura3 -52 his3 trp1 -289
This laboratory
supE44 DlacU169 (ø80lacZDM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1
Sambrook et al. (1989)
ApR URA3 ApR beg1 ApR beg1 ApR beg1 ApR URA3 EXG1 ApR EXG1 ApR URA3 EXG1 ApR URA3 PGK1PT ApR URA3 ADH2P -MFa1S -ADH2T ApR URA3 ADC1P -MFa1S -end1 ApR URA3 ADH2P -MFa1S -beg1 -ADH2T EXG1 ApR URA3 ADH2P -MFa1S -end1 -ADH2T ApR URA3 ADH2P -MFa1S -beg1 -ADH2T ApR URA3 ADH2P -MFa1S -end1 -ADH2T ADH2P -MFa1S -beg1 -ADH2T EXG1 ApR TRP1 FUR1 ApR fur1::LEU2
Hill et al. (1986) Hinchliffe (1984) This work This work This work This work This work Crous et al. (1995) La Grange et al. (1996) Van Rensburg et al. (1995) This work This work This work This work Kern et al. (1990) La Grange et al. (1996)
The constructs ADH2P -MFa1S -beg1 -ADH2T and ADH2P -MFa1S -end1 -ADH2T were designated BEG1 and END1, respectively.
(containing 1% yeast extract, 2% peptone and 2% glucose) and YPGE medium (containing 1% yeast extract, 2% peptone, 3% glycerol and 2% ethanol) were used to grow yeast cells. A laboratory strain of S. cere6isiae (Y294) was transformed with the recombinant plasmids and the transformants were grown in SC-Ura (containing 0.67% yeast nitrogen base without amino acids, 2% glucose and all required growth factors except uracil). Solid media contained 2% agar, unless otherwise indicated. Bacteria and yeasts were routinely cultured at 37°C and 30°C, respectively. Glucanase-producing yeast transformants were identified on YPD plates containing 0.3% medium viscosity carboxymethylcellulose (CMC; Sigma PC4888; Sigma, St. Louis, Mo., USA), 0.1% barley b-glucan (Sigma) or 0.4% lichenan (Sigma). The yeast transformants were grown for 2 days and the colonies were rinsed off the plates with TE buffer (10 mM Tris. HCl, 1 mM EDTA, pH 7) before staining the plates with 0.1% Congo red,
followed by destaining with 1 M NaCl in the case of carboxymethylcellulose (Teather and Wood, 1982). Colonies showing glucanase activity were identified by the formation of a clear zone around the colony.
2.3. Recombinant DNA and genetic techniques Standard methods were used for the isolation of plasmid DNA, genomic DNA and RNA, subcloning and manipulation of DNA fragments, and transformation of E. coli (Ausubel et al., 1987; Sambrook et al., 1989). Restriction endonucleases, T4 DNA ligase, Klenow (large) fragment of E. coli DNA polymerase I, DNase I and calf intestinal alkaline phosphatase were purchased from Boehringer Mannheim (Randburg, RSA) and used as recommended by the supplier. Nucleotide sequencing performed by the dideoxy chain-terminating method (Sanger et al., 1977) for double-stranded plasmid templates using a Seque-
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nase kit (United States Biochemical Corporation, Cleveland, Oh., USA). The [35S]-dATP used for the sequencing reactions was purchased from Amersham International (Buckinghamshire, UK). DNA amplification was performed by the polymerase chain reaction (PCR) technique (Innis and Gelfand, 1990; Saiki et al., 1988). Premixed PCR reagents (GeneAmp PCR reagent kit with Ampli Taq DNA polymerase) were purchased from Perkin-Elmer/Cetus (Norwalk, Ct., USA) and used according to the supplier’s recommendations. The following single-stranded oligodeoxyribonucleotides (restriction sites are underlined) were synthesized by the phosphoramidate method on a AutogenTM 650 DNA synthesizer: PBEG-5% (HindIII restriction site) 5%-GCCGAAGCTTTGCAAACAGGCGGATCGTTT-3% PBEG-3% (HindIII and NotI restriction sites) 5%-ACGTAAGCTTGCGGCCGCCATTATTTTTTTGTATAGCGCA-3%
2.4. Construction of a genomic library of S. cere6isiae Total genomic DNA from S. cere6isiae carrying the EXG1 exo-b-1,3-glucanase gene was partially digested with Sau3A. DNA fragments larger than 5 kb were recovered and inserted by ligation into the BamHI site of the E. coli-S. cere6isiae shuttle plasmid YEp24. A genomic library of 25 000 ApR (ampicillin resistant) clones, 90% of which were tetracycline sensitive (TcS), was obtained. Recombinant plasmid DNA prepared from the library was used to transform S. cere6isiae Y294 to Ura+. The Ura + transformants were screened for the EXG1 -encoded exo-b-1,3-glucanase activity by spraying SC-Ura plates with a solution containing 0.04% (w/v) 4-methyl-umbelliferyl-b-D-glucoside (MUG) in 50 mM acetate buffer (pH 5.2). After incubation at 37°C for 30 min, the plates were examined under UV light (254 nm). Transformants in which the exo-b-1,3-glucanase was overexpressed, appeared fluorescent because of the hydrolysis of MUG. A YEp24-based plasmid, pBG1, that conferred enhanced exo-b-1,3-glucanase activity on S. cere6isiae Y294, was recovered from one of the transformants and characterized by restriction analysis.
2.5. Southern and Northern blot hybridization Genomic DNA was isolated from S. cere6isiae, digested with NsiI and NcoI, separated on a 1% agarose gel and transferred to a nitrocellulose Hybond-N membrane (Amersham). Southern and Northern blot hybridizations were carried out as described by Sambrook et al. (1989). The presence of BEG1, END1 and EXG1 mRNA was confirmed by probing with an internal 720-bp HindIII DNA fragment of the beg1 gene, a 1563-bp HindIII DNA fragment of the B. fibrisol6ens end1 gene, and a 764-bp XbaI-BglII DNA fragment of the EXG1 gene, respectively. The DNA probes were labeled using a random primed DNA labeling kit (Boehringer Mannheim) and [a-32P]dATP (Amersham).
2.6. Yeast transformation S. cere6isiae Y294 was transformed with episomal-derived plasmid DNA by the lithium-acetate method of Gietz and Schiestl (1991). Plasmids pBEG, pEND and pBEE were maintained as autonomous circular minichromosomes in the yeast cells. The presence of these plasmids in the recombinant yeasts was verified by recovering them from the yeast cells, re-introducing them into E. coli and confirming their restriction endonuclease maps with those of the original plasmids.
2.7. Enzyme assays S. cere6isiae Y294, transformed separately with plasmid YEp352 and recombinant plasmids pBEG, pEND and pBEE, was cultured in 100 ml YPGE medium at 30°C. Samples were collected at different time points after inoculation and the same amount of cells was harvested from each of the samples. The culture supernatants, containing the secreted glucanase, were assayed with barley b-glucan (20 mg ml-1; Sigma) as substrate at 50°C. Appropriate dilutions of the supernatant were done using 0.05 M citrate-phosphate buffer (pH 6.2). For every 900 ml substrate 100 ml tant was added. Aliquots (1 ml) were removed in duplicate at intervals, heated (100°C) for 15 min
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and cooled to room temperature. Subsequently, 200 ml Congo red (100 mg ml-1) was added and the mixture diluted with buffer to 2 ml. Absorbancy was measured at 540 nm (Wood et al., 1988).
3. Results
3.1. Characterization of BEG1 Overlapping fragments of beg1 were subcloned from pPK5 into pUC18. The DNA sequence of the beg1 gene was determined by sequencing both strands. The nucleotide sequence of the 1.4-kb P6uI-ClaI fragment contained a single open reading frame (ORF) of 720 bp, encoding 240 amino acid residues (GenBank accession number U60830). The promoter region was located upstream of the ORF with a -35 sequence of TTCGAC followed by a 19-bp space and a -10 region of GATCAT. The deduced amino acid sequence from the B. subtilis NCIB 8565 beg1 -encoded protein (Beg1 ) was compared with those of endo-1,3-1,4-glucanases from five Bacillus species: Beg1 showed higher homology with BglA of B. amyloliquefaciens (97%; Hofemeister et al., 1986) than with BglS of B. subtilis (91%; Murphy et al., 1984). The homologies with BglL of B. licheniformis (Lloberas et al., 1991), GluM of B. marcerans (Borriss et al., 1990) and GluB of B. polymyxa (Gosalbes et al., 1991) were 84%, 76% and 75%, respectively (Fig. 1). The protein homology data suggest that Beg1 of B. subtilis NCIB 8565 and BglA of B. amyloliquefaciens are closely related even with regard to their leader peptide sequences. BglS of B. subtilis and BglL of B. licheniformis are more distantly related to Beg1 and BglA. However, GluM of B. marcerans and GluB of B. polymyxa differ more substantially from the other four endo-b-1,3-1,4-glucanases, particularly within their leader peptide sequences. These data correspond well with the morphological grouping of Bacillus species into three groups with B. amyloliquefaciens, B. subtilis and B. licheniformis within Group I and B. marcerans and B. polymyxa within Group II (Slepecky, 1992).
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3.2. Construction of BEG1, END1 and EXG1 A 3.5-kb EcoRI fragment from plasmid pEHB9 (Hinchliffe and Box, 1984), carrying the endo-b1,3-1,4-glucanase gene (beg1 ), was subcloned into pUC18, generating plasmid pPK4. The beg1 gene has been located on a 1.4-kb P6uI-ClaI fragment, contained within the 3.5-kb EcoRI DNA insert. The 1.4-kb P6uI-ClaI fragment from plasmid pPK4 was isolated, end-filled with Klenow enzyme and subcloned into the unique SmaI site of pUC18, generating recombinant plasmid pPK5. Based on the nucleotide sequence of beg1, oligodeoxynucleotides PBEG-5% and PBEG-3% were synthesized and used as primers to amplify a 720-bp HindIII fragment containing the beg1 gene. The resulting PCR product was subcloned into the HindIII site of the multicopy E. coli – S. cere6isiae shuttle plasmid pDLG4 (La Grange et al., 1996), thereby generating plasmid pBEG. In pBEG, the beg1 ORF was fused in-frame to the secretion signal sequence of the yeast mating pheromone a-factor (MFa1S ) and inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2P ) and terminator (ADH2T ). The ADH2P -MFa1S -beg1 -ADH2T construct was designated BEG1. Previously, the B. fibrisol6ens endo-b-1,4-glucanase gene (end1 ) was also fused in-frame to ADH2P -MFa1S and linked to ADH2T. Construct ADH2P -MFa1S -end1 -ADH2T (designated END1 ) was subcloned into the multicopy E. coli-S. cere6isiae shuttle plasmid, YEp352, resulting in plasmid pEND1 (Van Rensburg et al., 1996). A 3.95-kb BamHI-NheI fragment from plasmid pBG1 (YEp24 containing the EXG1 gene from S. cere6isiae) was subcloned into the BamHI and XbaI sites of pUC19, thereby generating plasmid pEX191. A 3.5-kb HindIII fragment was subcloned from pEX191 into the HindIII restriction site of YEp352, resulting in pEXG. This multicopy E. coli-S. cere6isiae shuttle plasmid contains the EXG1 gene with its native promoter and terminator sequences. A partial digestion of pBEG with BamHI produced a 2857-bp fragment containing theBEG1 construct. This fragment was cloned into the
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Fig. 1. The level of homology among the deduced amino acid sequences of endo-b-1,3-1,4-glucanases of the following Bacillus species: B. subtilis strain NCIB 2117, B. amyloliquefaciens (BglA), B. licheniformis (BglL), B. macerans (GluM) and B. polymyxa (GluB).
BamHI site of pEXG, generating plasmid pBEX. A 6357-bp P6uII DNA fragment containing the BEG1 and EXG1 constructs was subcloned from
pBEX into the unique SmaI site of pEND (Van Rensburg et al., 1996), thereby generating pBEE (Fig. 2).
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Fig. 2. Restriction maps of recombinant plasmids pBEG, pEND and pBEE. All three plasmids are multi-copy E. coli-S. cere6isiae shuttle vectors. The bacterial structural genes were inserted into expression-secretion cassettes (ADH2P -MFa1S and ADH1T). Plasmid pBEG (BEG1 ) contains the B. subtilis beg1 endo-b-1,3-1,4-glucanase gene, plasmid pEND (END1 ) contains the B. fibrisol6ens end1 endo-b-1,4-glucanase gene and plasmid pBEE (BEG1, END1 and EXG1 ) contains the end1 gene together with the beg1 and EXG1 genes. EXG1 encodes the main exo-b-1,3-glucanase in S. cere6isiae. Constructs ADH2P -MFa1S -beg1 -ADH2T and ADH2P -MFa1S -end1 -ADH2T ) were designated BEG1 and END1, respectively.
3.3. Expression of BEG1, END1 and EXG1 in autoselecti6e strains of S. cere6isiae Plasmids YEp352, pBEG (BEG1 ), pEND (END1 ) and pBEE (BEG1, END1 and EXG1 ) were transformed into S. cere6isiae Y294. Autoselective strains that would allow plasmid selection in uracil-containing medium were constructed according to the method of Loison et al. (1986). The wild-type FUR1 gene of S. cere6isiae strains Y294[YEp352], Y294[BEG1 ], Y294[END1 ] and Y-
294 [BEG1, END1 and EXG1 ] was replaced with a defective fur1 ::LEU2 allele by homologous recombination (Rothstein, 1983), using the 3.27-kb NcoI/NsiI linear DNA fragment from plasmid pDF1 (La Grange et al., 1996). The FUR1 gene from S. cere6isiae encodes uracil phosphoribosyltransferase which catalyzes the conversion of uracil into uridine 5%-phosphate in the pyrimidine salvage pathway (Kern et al., 1990). By disrupting this gene, fur1::LEU2 strains of S. cere6isiae Y294 were not able to utilize uracil from the culture medium and were therefore not viable, unless they
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Fig. 3. Southern blot analysis of fur1::LEU2 disruption in the autoselective S. cere6isiae Y294 transformants. Total DNA isolated from S. cere6isiae Y294 (lane 1 ), Y294[ fur1::LEU2 BEG1 ] (lane 2 ), Y294[ fur1::LEU2 END1 ] (lane3 ), and Y294[ fur1::LEU2 BEG1,END1 and EXG1 ] (lane 4 ) was digested with NcoI and NsiI, and probed with a [a-32P]-dATPlabeled NcoI/NsiI fragment of the FUR1 gene, derived from plasmid pPE (Kern et al. 1990).
possessed a URA3 -containing plasmid to synthesize uridine 5%-phosphate de novo. Leu + yeast transformants were selected on SC-Leu-Ura plates. The DNA-DNA hybridization by Southern blot analysis confirmed the integration of the defective fur1::LEU2 allele into the FUR1 gene (Fig. 3). The DNA-RNA hybridization by Northern blot analysis revealed the presence of the BEG1, END1 and EXG1 transcripts in S. cere6isiae Y294 containing pBEE (Fig. 4), the BEG1 transcript in Y294 carrying pBEG, and END1 transcript in
Fig. 5. Time course of extracellular glucanase activity produced by yeast transformants. The activity of the transformant Y294[ fur1::LEU2 BEG1 ] is depicted by (), Y294[ fur1::LEU2 END1 ] is depicted by (), Y294[ fur1::LEU2 BEG1 END1 EXG1 ] is depicted by ( ).
Y294 carrying pEND. The extracellular glucanase activities produced by the various S. cere6isiae Y294 transformants are shown in Fig. 5.
4. Discussion The results presented here describe the construction of autoselective, glucanolytic strains of S. cere6isiae by complementing the endogenous exo-b-1,3-glucanase and the cell-wall-bound endo-b-1,3-glucanase activities of the yeast with
Fig. 4. Northern blot analysis of BEG1 (A) END1 (B) and EXG1 (C) transcripts prepared from yeast cells. RNA samples were loaded as follows: (A) lane 1, Y294[YEp352]; lane 2, Y294[pBEG]; lane 3, Y294[pBEE], and (B) lane 1, Y294[YEp352]; lane 2, Y294[pEND]; lane 3, Y294[pBEE]; and (C) lane 1, Y294[YEp352]; lane 2, Y294[pBEE]. The nylon membrane was probed with internal fragments of the B. subtilis beg1, B. fibrisol6ens end1 and S. cere6isiae EXG1 genes. A RNA was used as a molecular size standard and the sizes (in kb) are indicated on the left.
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the B. subtilis endo-b-1,3-1,4-glucanase gene (beg1 ), the B. fibrisol6ens endo-b-1,4-glucanase gene (end1 ), and the over-expression of the S. cere6isiae exo-b-1,3-glucanase gene (EXG1 ). Transcription of both the BEG1 and END1 constructs was directed by the ADH2 promoter and termination sequences. The transcription of the EXG1 gene was directed by its native promoter and termination sequences. DNA-RNA hybridization by Northern blot analysis revealed the presence of the BEG1 transcript in S. cere6isiae transformant Y294[BEG1 ] carrying pBEG, the END1 transcript in Y294[END1 ] containing pEND, and the BEG1, END1 and EXG1 transcripts in Y294[BEG1 +END1 +EXG1 ] carrying pBEE (Fig. 1). The Northern blot in Fig. 4B clearly showed that transcription levels in the transformants containing plasmid pBEE were much lower than in the transformants containing pBEG or pEND. This is possibly caused by the titration of transcription factors or that the large size of pBEE caused the copy number of the episomal plasmid to decrease. Efficient secretion of the endo-b-1,4-glucanase and endo-b-1,3-1,4-glucanase from S. cere6isiae transformants was facilitated by in-frame fusions of the coding sequence for these mature enzymes to the MFa1 -encoded leader sequence. The 89amino acid MFa1 signal peptide, ending in the Lys-Arg-(Glu-Ala)2 hexapeptide (Kurjan and Herskowitz, 1982), has been used extensively for the secretion of various heterologous proteins by S. cere6isiae (Bitter et al., 1984, 1989; Brake, 1990). The native secretion signal sequence of the exo-1,3-glucanase mediated secretion in the S. cere6isiae transformants. The Northern blot analysis (Fig. 4) and results from time-course experiments (Fig. 5) indicated that not only was the transcription of the BEG1, END1 and EXG1 constructs efficiently promoted, but that the S. cere6isiae transformants also secreted biologically active endo-b-1,3-1,4-glucanase, endo-b-1,4-glucanase and/or exo-b-1,3-glucanase. It is relatively difficult to assay for endo-b-1,31,4-glucanase activity by measuring the amount of reducing sugars. The problem arises due to the fact that in barley b-glucan there are only a few 1,4-linkages next to 1,3-linkages, allowing for only
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a few places where the enzyme can cut the glucan chain. This results in the production of only a few reducing sugar ends and if measured by the dinotrosalicyclic acid (DNS) method (Miller et al., 1960) an effect is not detected. Interestingly, although only a few reducing sugar ends are formed, a large clearing zone is formed during the plate assay with Congo red. Results of the plate assays we performed clearly showed that the endo-b-1,3-1,4-glucanase only utilized barley bglucan and lichenan, but not CMC (results not shown), whereas the endo-b-1,4-glucanase hydrolyzed all three substrates. These results confirmed that endo-b-1,3-1,4-glucanases only hydrolyze b-1,4-linkages adjacent to b-1,3-linkages (Wolf et al., 1995), because CMC contains no 1,4-linkages next to 1,3-linkages. Based on the above results, we decided to use a liquid assay utilizing the breakdown of the glucan/Congo red complex (Wood et al., 1988). The data indicated that transformants containing plasmid pBEG (BEG1 ) generated a maximum of 16% breakdown of the glucan/Congo red complex in 10 h, whereas the transformants containing plasmid pEND (END1 ) produced 34% breakdown of the complex. Transformants carrying pBEE (BEG1, END1 and EXG1 ) produced a maximum breakdown of 37%. This clearly indicated that co-expression did not significantly improve the breakdown of the barley-b-glucan/Congo red complex in this specific time span, but the results did not show the size and type of the end products. Significantly different results could possibly have been obtained if a different type of glucan had been used which contains more 1,3-linkages or even 1,6-linkages (glucan produced by B. cinerea), since the exo-b-1,3-glucanase also contains 1,6-activity.
Acknowledgements The authors thank Drs E. Hinchliffe (Bass PLC, Burton-on-Trent) and D.R. Woods (University of Cape Town) for providing the B. subtilis endo-b-1,3-1,4-glucanase and B. fibrisol6ens endob-1,4-glucanase genes, respectively. We gratefully acknowledge the financial support from the Foun-
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dation for Research Development, Industrial Development Corporation of Southern Africa and the South African wine industry (Winetech).
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